Size-based genomic analysis

ABSTRACT

Systems, methods, and apparatuses for performing a prenatal diagnosis of a sequence imbalance are provided. A shift (e.g. to a smaller size distribution) can signify an imbalance in certain circumstances. For example, a size distribution of fragments of nucleic acids from an at-risk chromosome can be used to determine a fetal chromosomal aneuploidy. A size ranking of different chromosomes can be used to determine changes of a rank of an at-risk chromosome from an expected ranking. Also, a difference between a statistical size value for one chromosome can be compared to a statistical size value of another chromosome to identify a significant shift in size. A genotype and haplotype of the fetus may also be determined using a size distribution to determine whether a sequence imbalance occurs in a maternal sample relative to a genotypes or haplotype of the mother, thereby providing a genotype or haplotype of the fetus.

CROSS-REFERENCES TO RELATED APPLICATIONS

The present application claims priority from and is a non-provisionalapplication of U.S. Provisional Application No. 61/259,076, entitled“Detection of Chromosomal Imbalance” filed Nov. 6, 2009, and U.S.Provisional Application No. 61/360,399, entitled “Size-Based GenomicAnalysis” filed Jun. 30, 2010, the entire contents of which are hereinincorporated by reference for all purposes.

The present application is also related to U.S. application Ser. No.12/178,181, entitled “Diagnosing Fetal Chromosomal Aneuploidy UsingMassively Parallel Genomic Sequencing” filed Jul. 23, 2008; U.S.application Ser. No. 12/614,350, entitled “Diagnosing Fetal ChromosomalAneuploidy Using Genomic Sequencing With Enrichment,” and concurrentlyfiled U.S. application entitled “Fetal Genomic Analysis From A MaternalBiological Sample”, the entire contents of which are herein incorporatedby reference for all purposes.

BACKGROUND

The discovery of fetal DNA in maternal plasma in 1997 has opened up newpossibilities for noninvasive prenatal diagnosis (Lo et al Lancet 1997;350: 485-487). This technology has been rapidly translated to clinicalapplications, with the detection of fetal-derived, paternally-inheritedgenes or sequences, e.g. for fetal sex determination and for fetal RhDstatus determination. However, prenatal diagnostic applicationsinvolving genomic targets which are present in both the maternal andfetal genomes, e.g., chromosome 21, are much more challenging.

Recently, it has been demonstrated that single molecule countingtechniques, with their superior quantitative precision, might be apromising solution for this problem (Lo et al Proc Natl Acad Sci USA2007; 104: 13116-13121; Fan et al Anal Chem 2007; 79: 7576-7579; U.S.patent application Ser. No. 11/701,686; Chiu et al Trends Genet 2009;25: 324-331; Chiu et al Proc Natl Acad Sci USA 2008; 105: 20458-20463;Fan et al Proc Natl Acad Sci USA 2008; 105: 16266-16271). Such methodsachieve diagnostic goals through the observation of quantitativedifferences in the number of molecules from selected genomic locationsbetween disease and health. For example, for the diagnosis of fetal Downsyndrome, the number of molecules from chromosome 21 will be increasedwhen the fetus is suffering from trisomy 21 (Chiu et al Proc Natl AcadSci USA 2008; 105: 20458-20463; Fan et al Proc Natl Acad Sci USA 2008;105: 16266-16271).

However, such counting techniques may suffer from a limited number ofdata points or other disadvantages. Therefore, it is desirable toprovide new methods, systems, and apparatus for performing prenataldiagnosis having certain advantages over existing techniques.

BRIEF SUMMARY

Certain embodiments of the present invention can provide systems,methods, and apparatus can use a size-based analysis to perform aprenatal diagnosis of a sequence imbalance (e.g. a fetal chromosomalaneuploidy) in a biological sample obtained from a pregnant femalesubject. For example, a size distribution of fragments of nucleic acidmolecules for an at-risk chromosome can be used to determine a fetalchromosomal aneuploidy. Some embodiments can also detect other sequenceimbalances, such as a sequence imbalance in the biological sample(containing mother and fetal DNA), where the imbalance is relative to agenotype, mutation status, or haplotype of the mother. Such an imbalancecan be determined via a size distribution of fragments (nucleic acidmolecules) corresponding to a particular sequence relative to a sizedistribution to be expected if the sample were purely from the mother,and not from the fetus and mother. A shift (e.g. to a smaller sizedistribution) can signify an imbalance in certain circumstances.

In one embodiment, a ranking of a size distribution (e.g. a statisticalvalue representing a size distribution) of fragments from respectivechromosomes relative to each other are used to determine an imbalance.For instance, a ranking of the size of fragments of the at-riskchromosome in the test sample can be compared to the ranking for theat-risk chromosome that was obtained from a reference biological sample.A diagnosis can be performed based on the comparison. As an example, ifthe ranking changes (e.g. indicating a reduction in size of the nucleicacid fragments) by a specified amount then a diagnosis that a fetalchromosomal aneuploidy exists in the at-risk chromosome may be made. Invarious embodiments using such ranking analysis, all 22 autosomes andthe sex chromosomes can be used, or a subset of the chromosomes can beused.

In another embodiment, a difference between a size (e.g. a statisticalvalue representing a size distribution) of the fragments of the at-riskchromosome and a size of the fragments of a reference chromosome isused. For example, if the difference in the size is greater or smallerthan a cutoff (also called a threshold), then a diagnosis that a fetalchromosomal aneuploidy exists in the at-risk chromosome can be made.

According to one example embodiment, a method for performing prenataldiagnosis of a sequence imbalance in a biological sample obtained from apregnant female subject is provided. The biological sample includesnucleic acid molecules that are part of nucleic acid sequences. For eachof a plurality of the nucleic acid molecules in the biological sample, asize of the nucleic acid molecule is measured, and from which nucleicacid sequence the nucleic acid molecule derives is identified. Acomputer system determines a size distribution of the nucleic acidmolecules corresponding to a first sequence. Based on the determinedsize distribution, a classification of whether a sequence imbalanceexists for the first sequence is determined.

According to another example embodiment, a method for performingprenatal diagnosis of a fetal chromosomal aneuploidy in a biologicalsample obtained from a pregnant female subject is provided. For each ofa plurality of the nucleic acid molecules in the biological sample, asize of the nucleic acid molecule is measured, and from which chromosomethe nucleic acid molecule derives is identified. A computer systemcalculates a statistical value from the sizes of nucleic acid moleculescorresponding to a chromosome. A statistical value is calculated foreach of a plurality of chromosomes. A ranking of the chromosomes isdetermined based on the statistical value. The determined ranking of afirst chromosome is compared to another ranking of the first chromosomeobtained from a reference biological sample. Based on the comparison, aclassification of whether a fetal chromosomal aneuploidy exists for thefirst chromosome is determined.

According to another example embodiment, a method for performingprenatal diagnosis of a fetal chromosomal aneuploidy in a biologicalsample obtained from a pregnant female subject is provided. For each ofa plurality of the nucleic acid molecules in the biological sample, asize of the nucleic acid molecule is measured, and from which chromosomethe nucleic acid molecule derives is identified. A computer systemcalculates a first statistical value from the sizes of nucleic acidmolecules corresponding to a first chromosome. The computer systemcalculates a second statistical value from the sizes of nucleic acidmolecules corresponding to one or more second chromosomes. A separationvalue between the first statistical value and the second statisticalvalue is determined. The separation value is compared to one or morecutoff values. Based on the comparison, a classification of whether afetal chromosomal aneuploidy exists for the first chromosome isdetermined.

Other embodiments of the invention are directed to systems and computerreadable media associated with methods described herein. In oneembodiment, the computer readable medium contains instructions forreceiving data and analyzing data, but not instructions for directing amachine to create the data (e.g. sequencing nucleic acid molecules). Inanother embodiment, the computer readable medium does containinstructions for directing a machine to create the data. In oneembodiment, a computer program product comprises a computer readablemedium storing a plurality of instructions for controlling a processorto perform an operation for methods described herein. Embodiments arealso directed to computer systems configured to perform the steps of anyof the methods described herein, potentially with different componentsperforming a respective step or a respective group of steps.

Reference to the remaining portions of the specification, including thedrawings and claims, will realize other features and advantages ofembodiments of the present invention. Further features and advantages,as well as the structure and operation of various embodiments of thepresent invention, are described in detail below with respect to theaccompanying drawings. In the drawings, like reference numbers canindicate identical or functionally similar elements.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a flowchart illustrating a method 100 of performing prenataldiagnosis of a sequence imbalance in a biological sample obtained from apregnant female subject according to embodiments of the presentinvention.

FIG. 2 is a plot showing median rankings of chromosomes in terms of thesize of sequences aligned to it (when version 1 of the Illumina ClusterGeneration Reagent Kit was used) according to embodiments of the presentinvention.

FIG. 3 is a graph demonstrating that a size analysis of sequencesderived from different chromosomes in maternal plasma according toembodiments of the present invention can be used for the noninvasiveprenatal detection of fetal chromosomal 21 aneuploidies

FIG. 4 is a flowchart illustrating a method of performing prenataldiagnosis of a fetal chromosomal aneuploidy in a biological sampleobtained from a pregnant female subject using rankings of a statisticalvalue of size according to embodiments of the present invention.

FIG. 5 is a table illustrating comparisons of chromosome 21 tochromosome 7 and chromosome 14 in terms of the size of sequences alignedto them according to embodiments of the present invention.

FIG. 6 is a flowchart illustrating a method of performing prenataldiagnosis of a sequence imbalance in a biological sample obtained from apregnant female subject using a comparison of statistical value of asize of fragments for a genomic location according to embodiments of thepresent invention.

FIG. 7 shows a plot 700 of the fraction of total length contributed byshort fragments (F) against the cutoff size (w) according to embodimentsof the present invention.

FIG. 8 shows a plot 800 of the fraction of total length contributed byshort fragments (F) for chromosome 21 (solid line) and the referencechromosomes (all autosomes except chromosomes 13, 18 and 21) (dottedline) against the cutoff size for a euploid pregnancy according toembodiments of the present invention.

FIG. 9 shows a plot 900 of the F values for chromosome 21 (solid line)and the reference chromosomes (all autosomes except chromosomes 13, 18and 21) (dotted line) against the cutoff size for a trisomy 21 pregnancyaccording to embodiments of the present invention.

FIG. 10 shows a plot 1000 of ΔF_((21-Ref)) between chromosome 21 and thereference chromosomes (all autosomes except chromosomes 13, 18 and 21)against the size cutoff for a euploid and a trisomy 21 pregnancyaccording to embodiments of the present invention.

FIG. 11 shows a table 1100 for the gender and classification of 120pregnancies according to embodiments of the present invention.

FIG. 12 illustrates the ranking of the different chromosomes for foursamples of different disease or non-disease states according toembodiments of the present invention.

FIG. 13 shows a ranking of chromosome 13 for the 120 cases of euploid,trisomy 13, trisomy 18, and trisomy 21 according to embodiments of thepresent invention.

FIG. 14 shows a ranking of chromosome 18 for the 120 cases of euploid,trisomy 13, trisomy 18, and trisomy 21 according to embodiments of thepresent invention.

FIG. 15 shows a ranking of chromosome 21 for the 120 cases of euploid,trisomy 13, trisomy 18, and trisomy 21 according to embodiments of thepresent invention.

FIG. 16 is a table illustrating comparisons of chromosome 13 tochromosome 5 and chromosome 6 in terms of the size of sequences alignedto them according to embodiments of the present invention. Thecomparisons of chromosomes 5 and 6 with chromosome 13 are used for thedetection of changes in the size of chromosome 21 sequences in trisomy13 pregnancies. Results from euploid and trisomy 18 pregnancies areincluded for comparison.

FIG. 17 is a table illustrating comparisons of chromosome 18 tochromosome 12 and chromosome 14 in terms of the size of sequencesaligned to them according to embodiments of the present invention.Results from euploid and trisomy 13 pregnancies are included forcomparison according to embodiments of the present invention.

FIG. 18 shows a difference in the fractions of the total lengthcontributed by short fragments between chromosome 18 and the referencechromosomes (ΔF_((18-Ref))) at 150 bp according to embodiments of thepresent invention.

FIG. 19 shows a difference in the fractions of the total lengthcontributed by short fragments between chromosome 21 and the referencechromosomes (ΔF_((21-Ref))) at 150 bp according to embodiments of thepresent invention.

FIG. 20 shows a list of the GC contents of different chromosomes (NCBIbuild 36, version 48) according to embodiments of the present invention.

FIG. 21 shows a difference in the fractions of the total lengthcontributed by short fragments between chromosome 13 and the referencechromosomes (ΔF_((18-Ref))) at 150 bp according to embodiments of thepresent invention.

FIG. 22 is a plot showing correlation between the median size ofsequences aligned to chromosome 21 and percentage of sequences alignedto the Y-chromosome according to embodiments of the present invention.

FIG. 23A-23C are plots showing correlation between the median size ofsequences respectively aligned to chromosomes 18, 13, and 21 andpercentage of sequences aligned to the Y-chromosome according toembodiments of the present invention.

FIG. 24 shows a comparison of the accuracies of an embodiment of thepresent invention and another method for the noninvasive detection offetal aneuploidies (trisomy 13 and trisomy 18) using maternal plasma DNAanalysis.

FIGS. 25A-25C show diagrams for different scenarios for genotypes of apregnant woman and the fetus according to embodiments of the presentinvention.

FIG. 26 shows an example where the mother is heterozygous and the fatheris homozygous according to embodiments of the present invention.

FIG. 27 shows an example where the fetus inherits Hap I from the motherwhen the parental haplotype is as shown in FIG. 26 according toembodiments of the present invention.

FIG. 28 shows a table illustrating a size analysis for type αsingle-nucleotide polymorphisms (SNPs) on chromosome 22 according toembodiments of the present invention.

FIG. 29 shows a table illustrating a size analysis for type β SNPs onchromosome 22 according to embodiments of the present invention.

FIG. 30 shows a plot of ΔF_((Hap I-Hap II)) for type α and type β SNPson chromosome 22 according to embodiments of the present invention.

FIG. 31A is a table that provides a size analysis of plasma DNA withouttarget enrichment according to embodiments of the present invention.

FIG. 31B is a table that provides a size analysis of plasma DNA withtarget enrichment according to embodiments of the present invention.

FIG. 32 is a plot of ΔF for the T21 and euploid samples with and withouttarget enrichment according to embodiments of the present invention.

FIG. 33 shows a block diagram of an exemplary computer apparatus usablewith system and methods according to embodiments of the presentinvention.

DEFINITIONS

The term “biological sample” as used herein refers to any sample that istaken from a subject (e.g., a human, such as a pregnant woman) andcontains one or more nucleic acid molecule(s) of interest.

The term “nucleic acid” or “polynucleotide” refers to a deoxyribonucleicacid (DNA) or ribonucleic acid (RNA) and a polymer thereof in eithersingle- or double-stranded form. Unless specifically limited, the termencompasses nucleic acids containing known analogs of naturalnucleotides that have similar binding properties as the referencenucleic acid and are metabolized in a manner similar to naturallyoccurring nucleotides. Unless otherwise indicated, a particular nucleicacid sequence also implicitly encompasses conservatively modifiedvariants thereof (e.g., degenerate codon substitutions), alleles,orthologs, SNPs, copy number variants, and complementary sequences aswell as the sequence explicitly indicated. Specifically, degeneratecodon substitutions may be achieved by generating sequences in which thethird position of one or more selected (or all) codons is substitutedwith mixed-base and/or deoxyinosine residues (Batzer et al., NucleicAcid Res. 19:5081 (1991); Ohtsuka et al., J. Biol. Chem. 260:2605-2608(1985); and Rossolini et al., Mol. Cell. Probes 8:91-98 (1994)). Theterm nucleic acid is used interchangeably with gene, cDNA, mRNA, smallnoncoding RNA, micro RNA (miRNA), Piwi-interacting RNA, and shorthairpin RNA (shRNA) encoded by a gene or locus.

The term “gene” means the segment of DNA involved in producing apolypeptide chain or transcribed RNA product. It may include regionspreceding and following the coding region (leader and trailer) as wellas intervening sequences (introns) between individual coding segments(exons).

The term “clinically relevant nucleic acid sequence” (also referred toas a target sequence or chromosome) as used herein can refer to apolynucleotide sequence corresponding to a segment of a larger genomicsequence whose potential imbalance is being tested or to the largergenomic sequence itself One example is the sequence of chromosome 21.Other examples include chromosome 18, 13, X and Y. Yet other examplesinclude mutated genetic sequences or genetic polymorphisms or copynumber variations that a fetus may inherit from one or both of itsparents, or as a de novo mutation in the fetus. In some embodiments,multiple clinically relevant nucleic acid sequences, or equivalentlymultiple markers of the clinically relevant nucleic acid sequence, canbe used to provide data for detecting the imbalance. For instance, datafrom five non-consecutive sequences on chromosome 21 can be used in anadditive fashion for the determination of possible chromosomal 21imbalance, effectively reducing the needed sample volume to ⅕.

The term “reference nucleic acid sequence” as used herein refers to anucleic acid sequence whose size distribution is used to compare againstthe target sequence. Examples of a reference nucleic acid sequenceinclude a chromosome, a part of a chromosome, a particular allele (e.g.of a mother), a particular haplotype, a genome, or an artificiallysynthesized nucleic acid sequence. Such reference nucleic acid sequencescan either exist endogenously in the sample, or added exogenously duringsample processing or analysis. In some embodiments, the referencenucleic acid sequences demonstrate a size profile that is representativeof a healthy state without disease.

The term “based on” as used herein means “based at least in part on” andrefers to one value (or result) being used in the determination ofanother value, such as occurs in the relationship of an input of amethod and the output of that method. The term “derive” as used hereinalso refers to the relationship of an input of a method and the outputof that method, such as occurs when the derivation is the calculation ofa formula.

The term “parameter” as used herein means a numerical value thatcharacterizes a quantitative data set and/or a numerical relationshipbetween quantitative data sets. For example, a ratio (or function of aratio) between a first amount of a first nucleic acid sequence and asecond amount of a second nucleic acid sequence is a parameter.

As used herein, the term “locus” or its plural form “loci” is a locationor address of any length of nucleotides (or base pairs) which has avariation across genomes.

The term “sequence imbalance” as used herein means any significantdeviation as defined by at least one cutoff value in a quantity of theclinically relevant nucleic acid sequence from a reference quantity. Asequence imbalance can include chromosome dosage imbalance, allelicimbalance, mutation dosage imbalance, copy number imbalance, haplotypedosage imbalance, and other similar imbalances. As an example, anallelic or mutation dosage imbalance can occur when a fetus has adifferent genotype from the mother, thereby creating an imbalance at aparticular locus in the sample.

The term “chromosomal aneuploidy” as used herein means a variation inthe quantitative amount of a chromosome from that of a diploid genome.The variation may be a gain or a loss. It may involve the whole of onechromosome or a region of a chromosome.

The term “haplotype” as used herein refers to a combination of allelesat multiple loci that are transmitted together on the same chromosome orchromosomal region. A haplotype may refer to as few as one pair of locior to a chromosomal region, or to an entire chromosome. The term“alleles” refers to alternative DNA sequences at the same physicalgenomic locus, which may or may not result in different phenotypictraits. In any particular diploid organism, with two copies of eachchromosome (except the sex chromosomes in a male human subject), thegenotype for each gene comprises the pair of alleles present at thatlocus, which are the same in homozygotes and different in heterozygotes.A population or species of organisms typically includes multiple allelesat each locus among various individuals. A genomic locus where more thanone allele is found in the population is termed a polymorphic site.Allelic variation at a locus is measurable as the number of alleles(i.e., the degree of polymorphism) present, or the proportion ofheterozygotes (i.e., the heterozygosity rate) in the population. As usedherein, the term “polymorphism” refers to any inter-individual variationin the human genome, regardless of its frequency. Examples of suchvariations include, but are not limited to, single nucleotidepolymorphism, simple tandem repeat polymorphisms, insertion-deletionpolymorphisms, mutations (which may be disease causing) and copy numbervariations.

DETAILED DESCRIPTION

Fetal DNA molecules that are present in the plasma of pregnant womenhave been found to be generally shorter than maternally-derivedmolecules (Chan et al Clin Chem 2004; 50: 88-92; Li et al Clin Chem2004; 50: 1002-1011; US Patent Application 20050164241). Certainembodiments of the present invention can determine from the maternalplasma DNA whether the fetus has over- or under-representation of aparticular part of the genome by a change of the size of DNA moleculesderived from that part of the genome. As fetal DNA represents a minorfraction of DNA in maternal plasma, the overall degree of size change inmaternal plasma is likely to be small, and thus can be difficult todetect. In some embodiments, the sizes of a number of molecules aremeasured to arrive at a statistically significant difference betweendisease and health.

I. Determining Size of a Fragment

One method with which the sizes of a number of DNA molecules can bemeasured is by massively parallel genomic sequencing. This can beperformed for example by the Illumina Genome Analyzer platform (usingsequencing by synthesis) (Bentley D R et al Nature 2008; 456: 53-59),the ABI SOLiD (using sequencing by ligation) (McKernan et al Genome Res2009; 19: 1527-1541), the Roche 454 platform (Marguelis et al Nature2005; 437:376-380) and the Helicos single molecule sequencing platform(Harris et al Science 2008; 320: 106-109). It is also expected thatother massively parallel sequencing platforms can also be used, e.g. thePacific Biosciences (single molecule, real-time (SMRT™) technology) (Eidet al Science 2009; 323: 133-138), nanopore sequencing (Clarke Jet al.Nat Nanotechnol 2009; 4: 465-470), semiconductor sequencing (e.g. by IonTorrent (iontorrent.com), etc.

One way to obtain the DNA size information from such sequencing is toperform paired-end (PE) sequencing, in which both ends of a DNA moleculeare sequenced. Then, the sequences corresponding to both ends of themolecule can be mapped back to the reference genome (e.g. a referencehuman genome or a reference horse genome, or the genome of any animal ofinterest). In one embodiment, both ends are each sequenced at a lengththat is long enough to be mapped back, individually for each end, to thereference human genome (e.g. about 10-24 bases or 25-36 bases). Inanother embodiment, only a proportion of sequences can be mapped backwithout mismatch to the non-repeat region of the human genome. In oneaspect, the mapping may be unambiguuous if both sequences together areused in the mapping. In this scenario, even though each of the endsmight be too short to be mapped back with confidence, using bothsequences can provide unambiguous mapping. The size of the molecule canbe worked out by subtraction of the genomic coordinates of the ends ofthe two sequences.

In another embodiment, the size of the molecule can be obtained by acomplete, or close to complete, sequencing of the whole DNA molecule,instead of just the two ends. This can be done efficiently by sequencingplatforms with relatively long read-lengths, such as the Roche 454platform, the Pacific Biosciences single molecule, real-time (SMRT™)technology, and the Ion Torrent technology (iontorrent.com).

The throughput of the above-mentioned sequencing-based methods can beincreased with the use of indexing or barcoding (Cronn et al. NucleicAcids Res 2008; 36: e122). Thus, a sample or patient-specific index orbarcode can be added to nucleic acid fragments in a particular nucleicacid sequencing library. Then, a number of such libraries, each with asample or patient-specific index or barcode, are mixed together andsequenced together. Following the sequencing reactions, the sequencingdata can be harvested from each sample or patient based on the barcodeor index. This strategy can increase the throughput and thus thecost-effectiveness of the current invention.

In another embodiment, the nucleic acid molecules in the biologicalsample can be selected or fractionated prior to size analysis. In onevariant, the nucleic acid molecules are treated with a device (e.g. amicroarray or a solution containing probes) which would preferentiallybind nucleic acid molecules from selected loci in the genome (e.g. oneof chromosomes 21, 18, 13, or X), then the size analysis can beperformed on the bound subset of the nucleic acid molecules. In such anembodiment, a Nimblegen sequence capture system(nimblegen.com/products/seqcap/index.html) or an Agilent SureSelectTarget Enrichment System(opengenomics.com/SureSelect_Target_Enrichment_System), or similarplatforms can be used. In another embodiment, the unbound nucleic acidsubset can be differentially removed or degraded or digested

At least some embodiments can work with any single molecule analysisplatform in which the chromosomal origin and the length of the moleculecan be analyzed, e.g. electrophoresis, optical methods (e.g. opticalmapping and its variants, wikipediaOptical_mapping#cite_note-Nanocoding-3, and Jo et al. Proc Natl Acad SciUSA 2007; 104: 2673-2678), fluorescence-based method, probe-basedmethods, digital PCR (microfluidics-based, or emulsion-based, e.g.BEAMing (Dressman et al. Proc Natl Acad Sci USA 2003; 100: 8817-8822),RainDance (raindancetech.com/technology/per-genomics-research.asp)),rolling circle amplification, mass spectrometry, melting analysis (ormelting curve analysis), molecular sieving, etc. As an example for massspectrometry, a longer molecule would have a larger mass (an example ofa size value).

In one example, plasma DNA molecules were randomly sequenced by theIllumina Genome Analyzer system using a paired-end sequencing protocol.In this experiment, version 1 of the Illumina Paired-end (PE) ClusterGeneration Reagent Kit was used. Each end was sequenced for 36 bp. Thetwo ends of each sequence were aligned to the repeat-masked human genome(NCBI Build 36, version 48) using the eland_pair program in theGAPipeline-1.0 software package provided by Illumina. Only 32 bp out ofthe 36 bp from each end were used for alignment purposes.

In some embodiments, the PE reads meeting the following criteria can beused for subsequent analysis: (1) the individual members of eachsuggested pair were both sequenced on the same cluster position on thesequencing flow cell and could be aligned to the same chromosome withthe correct orientation as expected for the human reference genome; (2)the sequenced reads of both members of the pair could be aligned to therepeat-masked human reference genome without any nucleotide mismatch;(3) the sequenced reads of each member of the pair had a uniquenessscore >4; and (4) pairs demonstrating an insert size less than 600 bp.The size of each aligned sequence was then calculated according to theposition of each of the two ends.

II. Using Size Distribution to Determine Aneuploidy Status

FIG. 1 is a flowchart illustrating a method 100 of performing prenataldiagnosis of a sequence imbalance in a biological sample obtained from apregnant female subject according to embodiments of the presentinvention. While method 100 is mainly described with respect toanalyzing a fetal chromosomal aneuploidy, other embodiments of method100 and other methods herein can be applied to other sequence imbalances(e.g. identification of genotype or mutations). Method 100 and othermethods mentioned herein, may be performed wholly or partially by acomputer system including one or more processors.

Method 100 and any of the methods described herein may be totally orpartially performed with a computer system including a processor, whichcan be configured to perform the steps. Thus, embodiments are directedto computer systems configured to perform the steps of any of themethods described herein, potentially with different componentsperforming a respective step or a respective group of steps. Althoughpresented as numbered steps, steps of methods herein can be performed ata same time or in a different order. Additionally, portions of thesesteps may be used with portions of other steps from other methods. Also,all or portions of a step may be optional. Additionally, any of thesteps of any of the methods can be performed with modules, circuits, orother means for performing these steps.

In step 110, a size of at least some of the nucleic acid molecules (e.g.DNA or RNA) in the biological sample is measured. The nucleic acidmolecules are also referred to as fragments, as they are a fragment ofthe entire genome. The size may be measured via any suitable method, forexample, methods described above.

Step 120 identifies a location in a genome from which each of thenucleic acid molecules is derived. The location can be any part of agenome, which is human for the examples provided, but could be for othergenomes. For example, the location can be which chromosome number thefragment is derived, which part of a chromosome as may be defined bygenomic coordinates (e.g. a specific coordinate or range ofcoordinates), and even may be which one of the two chromosomes (assumingeuploid) the fragment is derived (originated).

In one embodiment, this identification can be performed by sequencingand comparing the sequence information with the reference human genomesequence. In another embodiment, this identification can be performed byhybridization to a panel of probes with known chromosomal origin. Theprobes could be labeled with one or more fluorescence labels, in eithera microarray format or in solution. In yet another embodiment, thenucleic acid molecules could be captured by a panel of probes, either insolution or on a solid surface, and then the captured (or the remainingnon-captured) nucleic acid molecules are sequenced. In some embodiments,where a sequence imbalance other than a chromosomal aneuploidy is to beidentified, a step of identifying from which chromosome that a fragmentis originated may be optional.

In step 130, a size distribution of the nucleic acid moleculescorresponding to a first location (e.g. a first chromosome) isdetermined. Various embodiments can use a variety of size distributions.In some embodiments, a size distribution relates to the rankings of thesizes (e.g., an average, median, or mean) of fragments of one chromosomerelative to fragments of other chromosomes. In other embodiments, a sizedistribution can relate to a statistical value of the actual sizes ofthe fragments of a chromosome. In one implementation, a statisticalvalue can include any average, mean, or median size of fragments of achromosome. In another implementation, a statistical value can include atotal length of fragments below a cutoff value, which may be divided bya total length of all fragments, or at least fragments below a largercutoff value.

In step 140, a classification of whether a sequence imbalance (e.g. afetal chromosomal aneuploidy) exists for the first location isdetermined based on the determined size distribution. In one embodiment,the ranking of a chromosome is compared to a reference ranking (e.g. oneof a euploid sample). If the change is significant (e.g. exceeding athreshold), then the sample can be classified as aneuploid. In anotherembodiment, a statistical value of the actual sizes are compared betweentwo chromosomes, or between groups of chromosomes. For example, adifference can be taken between the respective statistical values, andthe difference compared to a cutoff.

II. Size Distribution Rank

Embodiments can use values of the size of nucleic acid fragments of asample to determine if a chromosomal imbalance exists. The fragments arealso called sequences after a sequencing has been performed. In oneembodiment, a distribution of the size of fragments for a plurality ofchromosomes is determined, and the chromosomes are ranked based on astatistical value (e.g. average, mean, or median) of the distribution.For convenience, the term “size” can be used herein synonymously with astatistical value of size. It should be clear when the term “size” isreferring to the size of a specific fragment and a statistical measureof size of a set of fragments.

A. Rank

FIG. 2 is a plot 200 that illustrates the measured size distribution ofthe DNA fragments originated from different chromosomes in maternalplasma. As the measured size can reflect not just the DNA fragment sizein vivo, but also the contribution of the analytical steps, it isexpected that the size distribution might vary from platform to platform(e.g. for the Illumina Genome Analyzer and for the ABI SOLiD platform)and might even vary when different versions of reagents are used for aparticular platform. However, as long as the reference samples and thetest samples are analyzed using the same platform or reagent types,embodiments can be used in a platform/reagent-independent manner. Someembodiments may also be used with different platforms and/or reagenttypes, e.g., if any errors can be determined and corrected, or if theplatforms and/or reagent types can be shown to have closely matchedanalytical performances.

In FIG. 2, the 22 autosomes and chromosome X were compared in terms ofthe size of fragment sequences aligned to them. On the Y-axis, the rankis in the descending order of the size of sequences, i.e. the chromosomewith the longest sequences ranks 1 and the chromosome with the shortestsequences ranks 23. In one embodiment, the comparison (Kruskal-WallisOne Way Analysis of Variance on Ranks followed by Bonferroni-correctedpairwise comparisons) was made non-parametrically using the SigmaStat(SPSS) software. The comparison can be of any statistical valuerepresenting size, including a ranking of each of the individual sizesand a statistical analysis of the individual rankings for the sequencesof each chromosome. In one implementation, ties and skips in the ranknumbering are allowed.

In some embodiments, each of the sequences are mapped to a particularchromosome. Then, for each chromosome, one or more statistical valuesare determined for the sequences mapped to that chromosome. That isdifferent types of statistical values (e.g. mean and median) can becalculated for the sizes of each chromosome. The correspondingstatistical values can then be ranked. For example, the average sizesfor each chromosome can be compared to each other. If more than onestatistical value is used, then the statistical values can be combined(e.g. according to some formula, such as a weighted average), and thiscombined statistical value can be ranked. In one embodiment, therankings of the statistical values for a particular chromosome can becombined (e.g. as mentioned above for the statistical values), and thenthe combined rankings can be compared to each other.

In other embodiments, all of the sequences are individually rankedaccording to size. That is if there are 1,000,000 sequences, then therankings go from 1 to one million, with possible ties and skips in therank numbering. The rankings for all sequences mapped to the samechromosome can then be added together. The sum of the rankings can bedivided by the number of sequences aligned to the particular chromosometo arrive at the mean sequence ranking for the chromosome. Thechromosome with the highest mean sequence ranking can be marked as thelongest (ranks 1, highest on the Y-axis) and the chromosome with thelowest mean sequence ranking would be the shortest (ranks 23, lowest onthe Y-axis). In another embodiment, a median rank can be determined. Themedian ranks of the cases with euploid male fetus, euploid female fetusand trisomy 21 male fetus are shown in FIG. 2.

On the X-axis, the chromosomes are arranged in a descending order of thesize distribution of DNA fragments derived from each chromosome (withthe exception of the X chromosome (see later)). In one embodiment, forsuch ranking, only the euploid cases were used. The chromosome with thelongest measured size (e.g. length) is arranged on the left side. The Xchromosome has been placed on the right of the diagram because itsranking is governed by the sex of the fetus.

As mentioned above, the measured size might vary from platform toplatform (e.g., when one changes from the Illumina system to anothersystem). Thus, in one aspect, the size can refer to the ‘measured’ size,as opposed to an actual size. The size might even change when oneswitches from one version of the Illumina kit to another one, e.g. whenone changes from a version 1 paired-end cluster generation kit toversion 2. In one embodiment, users can perform a ranking for theirparticular system.

It can be seen from FIG. 2 that the mean rank of the X chromosome islower (i.e. becomes shorter) in the plasma of a pregnant woman carryinga female fetus, when compared with one carrying a male fetus. Theexplanation of this observation is that the DNA fragments released by afetus are shorter than those from the mother. Thus, by releasing adouble dose of the X chromosome, a female fetus will reduce the overallmeasured size fragments derived from the X chromosome in maternalplasma. Conversely, a male fetus would only be able to release a singledose of the X chromosome.

Also, one can see from FIG. 2 that the rank of chromosome 21 decreases(i.e. becomes shorter) in the plasma of a pregnant woman carrying atrisomy 21 fetus, when compared with one carrying a euploid fetus. Theexplanation of this observation can once again be traced back to themeasured size fragments derived from chromosome 21 in maternal plasma.Conversely, a euploid fetus would only be able to release two doses ofchromosome 21 per fetal cell.

FIG. 3 is a graph 300 demonstrating that a size analysis of sequencesderived from different chromosomes in maternal plasma according toembodiments of the present invention can be used for the noninvasiveprenatal detection of fetal chromosome 21 aneuploidies. In this example,the chromosome size is represented by a size rank when compared withother chromosomes. Thus, a larger size rank number indicates that achromosome has shorter DNA fragment sizes in maternal plasma.

Graph 300 demonstrates that the size rank number of chromosome 21 inmaternal plasma is larger (thus indicating shorter DNA fragments) forpregnancies involving a trisomy 21 fetus (ranging from rank 18 to 21),than when the fetus is euploid (ranging from rank 9 to 18). Theexplanation of this observation is that fetal DNA is shorter thanmaternal DNA and that the additional dose of chromosome 21 from atrisomy 21 fetus would lead to an overall shortening of a statisticalvalue of chromosome 21 sequences in maternal plasma.

B. Methods Using Rank

FIG. 4 is a flowchart illustrating a method of performing prenataldiagnosis of a fetal chromosomal aneuploidy in a biological sampleobtained from a pregnant female subject using rankings of a statisticalvalue of size according to embodiments of the present invention.

In step 410, a size of each of a plurality of nucleic acid molecules(fragments) obtained from the biological sample is measured. Note thatthis plurality of nucleic acid molecules may be a subset of all of thenucleic acid molecules obtained. The plurality of nucleic acid moleculesmay even be a subset of all the nucleic acid molecules that aresequenced, when sequencing is done as part of the size measurement.

In step 420, it is identified from which chromosome each of theplurality of nucleic acid molecules is derived. In various embodiments,the order of steps 410 and 420 can be reversed or occur simultaneously.For example, in the paired-end sequencing context, the genomic alignmentof a sequence can provide its chromosomal location, plus its length (bysubtraction of the start and end genomic coordinates). In oneembodiment, the chromosome can be identified as in step 120.

In step 430, for each of a plurality of chromosomes, a statistical valueis calculated from the sizes of nucleic acid molecules corresponding tothe chromosome. The statistical value can be calculated in any of theways described herein. For example, the statistical value could includea result of an initial ranking stage, as described above. In oneembodiment, only a portion of the nucleic acid molecules correspondingto any particular chromosome may be used.

In step 440, a ranking of the chromosomes is determined based on thestatistical values. In one embodiment, the ranking can be determinedusing a basic sorting algorithm. In another embodiment, more complicatedcomparison may be performed, such as a Kruskal-Wallis one way analysisof variance on ranks followed by Bonferroni-corrected pairwisecomparisons, or other suitable methods. In various embodiments, therankings could be integers, fractions, real numbers (e.g. in a range),or alphanumeric rankings based on a rubric (e.g. A-X).

In step 450, the determined ranking of a first chromosome is compared toanother ranking of the first chromosome obtained from a referencebiological sample. In one embodiment, the comparison is the comparisonof the determined ranking against a cutoff threshold (e.g. a singlevalue or range), which is determined from rankings of one or morereference biological samples. Such a cutoff value could be if the rankis 18 (or 19) or higher, as can be determined from FIG. 3. In anotherembodiment, a difference in the ranking of the first chromosome betweenthe two samples can be determined, and the difference can be compared toa cutoff. In one implementation, the reference biological sample hasbeen analyzed to determine that it does not contain the disease ofinterest, and it may even be determined that the sample does not containany relevant disease that might cause problems with a ranking.

In step 460, based on the comparison, a classification of whether afetal chromosomal aneuploidy exists for the first chromosome isdetermined. In one embodiment, the classification can be a binaryclassification of disease or not disease. In another embodiment, theclassification can be ternary in that an indeterminate classificationmay be used. In yet another embodiment, the classification can include aprobability of a particular classification, and thus effectively havemore than just three classifications.

III. Size Distribution Statistical Values of Size

In other embodiments, the size distribution can include a statisticalvalues of the size of the fragments (e.g. statistical values of theactual or absolute values for a particular genomic location), as opposedto the rankings In one embodiment, actual sizes of a first chromosomecan be compared to actual sizes of one or more reference chromosomes fora same test sample. For example, a separation value (e.g. a differenceor ratio) between these actual sizes of the first chromosome and the oneor more reference chromosomes can be compared to a cutoff In oneimplementation, the cutoff can be determined from reference samples. Inanother embodiment, a separation value between the actual sizes of thefragments of a chromosome between a test sample and a referencebiological sample might be used along with a cutoff In yet anotherembodiment, the actual size of fragments of a chromosome might becompared against a cutoff to obtain a reliable classification.

A. Absolute Size

Some examples demonstrate that one can achieve the noninvasive prenataldetection of trisomy 21 by comparing the absolute sizes of fragmentsderived from chromosome 21 with those of fragments derived from one ormore reference chromosomes. In one embodiment, chromosomes 7 and 14 canbe selected as reference chromosomes, as they can have size values (e.g.absolute size or size rankings) in maternal plasma that are relativelyclose to chromosome 21. In practice, the reference chromosome may be anychromosome that has a fragment size that is consistent in its relation,e.g., on a specific analytical platform and/or reagent type, tochromosome 21 (or other chromosome of interest) for euploid samples.

FIG. 5 is a table 500 illustrating comparisons of chromosome 21 tochromosome 7 and chromosome 14 in terms of the size of sequences alignedto the respective chromosomes according to embodiments of the presentinvention. The data for table 500 is obtained from 16 test samples. Foreach sample, a mean size is shown for fragments of each of chromosomes7, 14, and 21. A difference between the mean values is also provided. Ap value shows a likelihood that each of the differences would occur in ahealthy sample.

As can be seen from table 500 of FIG. 5, the sequences aligned tochromosome 21 were significantly shorter (e.g. by mean size) than thesequences aligned to chromosome 7 and chromosome 14 for all the trisomy21 pregnancies (Mann-Whitney rank-sum test, p-value <0.001). Shorteningat this degree of statistical significance was not observed in theeuploid pregnancies. Thus, table 500 indicates that for all of thetrisomy 21 pregnancies, the difference of the mean fragment size betweenchromosome 21 and chromosome 7 was greater than 1 bp whereas none of theeuploid cases showed a difference more than 1 bp. Accordingly, 1 bp canprovide an accurate cutoff for determining a classification. Similarly,for all of the trisomy 21 pregnancies, the mean fragment size fromchromosome 14 is consistently larger than that of chromosome 21. Indeed,if a cutoff of 0.5 bp is used for the observed ‘lengthening’ ofchromosome 14 fragments when compared with those from chromosome 21, alltrisomy 21 cases can be distinguished from the non-trisomy 21 cases.Thus, in one embodiment, a cutoff value can be determined from one ormore reference samples.

B. Methods Using Absolute Size

FIG. 6 is a flowchart illustrating a method of performing prenataldiagnosis of a sequence imbalance in a biological sample obtained from apregnant female subject using a comparison of statistical value of asize of fragments for a genomic location according to embodiments of thepresent invention. In one aspect, method 600 can be directed todetermining a classification of a sequence imbalance based on aseparation value (e.g. a difference or ratio) for the size of fragmentsof a first chromosome and the size of fragments of one or more referencechromosomes.

In step 610, a size of a plurality of nucleic acid molecules obtainedfrom the biological sample is measured. Note that the plurality ofnucleic acid molecules may be obtained and include similar fragments asnoted for step 410.

In step 620, a genomic location is identified from which each of thenucleic acid molecules is derived. The location can be any part of agenome, as is described for step 120 and elsewhere. For example, it isidentified which chromosome each of the plurality of nucleic acidmolecules is derived. This determination can be made by a mapping to areference genome, as is described herein.

In step 630, a first statistical value is calculated from the sizes ofthe nucleic acid molecules corresponding to a first genomic location(e.g. a first chromosome). In one embodiment, the first statisticalvalue can be an average, mean, or median size of the fragmentscorresponding to the first chromosome. In another embodiment, the firststatistical value can include a sum of the length of fragments below afirst size, which can be a type of cutoff. For example, each of thefragments that are smaller than 200 bp can have their lengths summed.The sum can be divided by another number, such as a sum of the lengthsof all fragments corresponding to the first chromosome or a sum of thelengths of fragments greater than a second size cutoff (which may be thesame as the first size). For example, the first statistical value can bea ratio of the total length of fragments below a first size cutoffrelative to a total length of fragments, or a ratio of the total lengthof small fragments relative to a total length of large fragments.

In step 640, a second statistical value is calculated from the sizes ofthe nucleic acid molecules corresponding to a second genomic location(e.g. a second chromosome). The second chromosome can be considered areference chromosome. In one embodiment, a statistical value for aplurality of reference chromosomes can be calculated. In oneimplementation, the statistical values can be combined such that thestatistical value could be of one or more second chromosomes. In anotherembodiment, the statistical values for the plurality of referencechromosomes may be compared to individually, as mentioned below.

In step 650, the first statistical value and the second statistical arecompared to obtain a separation value. In one embodiment, the separationvalue can be a difference between the first statistical value and thesecond statistical value is determined. In another embodiment, theseparation value can be a ratio of the two statistical values. In yetanother embodiment, a plurality of separation values can be determined,e.g., one for each second statistical value, which can be calculated foreach reference chromosome.

In step 660, the separation value is compared to one or more cutoffvalues. In one embodiment, the comparison can be performed for each of aplurality of separation values. For example, as mentioned above, adifferent separation value can be determined between the firststatistical value and each second separation value. In variousimplementations, each separation value can be compared to the same ordifferent cutoff values. In another embodiment, a separation value iscompared to two cutoff values to determine whether the separation valueis within a particular range. The range can include one cutoff todetermine if a non-normal data point occurs (e.g. an aneuploidy) and asecond cutoff could be used to determine if the data point is likelycaused by an error in measurement or analysis (e.g., if the separationvalue is larger than ever would be expected, even for a diseasedsample).

In step 670, a classification of whether a sequence imbalance (e.g. afetal chromosomal aneuploidy) exists for the first genomic location isdetermined based on the comparison. In one embodiment, a plurality ofcutoffs (e.g. N cutoffs) can be used for a single separation value. Insuch an embodiment, N+1 classifications can be determined. For example,two cutoffs may be used to determine the classifications of euploid(normal or healthy), indeterminate, and aneuploid (diseased orunhealthy). In another embodiment where a plurality of comparisons areperformed (e.g. one for each separation value), the classification canbe based on each of the comparisons. For example, a rule based methodcan look at the classifications resulting from each of the comparisons.In one implementation, a definitive classification is only provided whenall of the classifications are consistent. In another implementation,the majority classification is used. In yet another implementation, amore complicated formula may be used based on how close each of theseparation values is to a respective cutoff value, and these closenessvalues can be analyzed to determine a classification. For example, thecloseness values could be summed (along with other factors, such as anormalization) and the result could be compared to another cutoff value.

In other embodiments, variations of method 600 can also be applied to adirect comparison of a statistical value for the first chromosome to acutoff value, which can be derived from a reference sample. Someembodiments can also be used for analyzing biological samples fromnon-pregnant individuals. Such an analysis can just look at astatistical value for a size of all of the fragments of a sample andcompare the statistical value or a separation value to a cutoff todetermine whether a sequence imbalance might exist. If an imbalance isclassified as existing, further analysis of a location of the imbalancecan be performed, e.g., by analyzing statistical size values and/orseparation values for a particular genomic location (e.g. a chromosome).

C. Using Total Lengths of Short Fragments

As mentioned above, in some embodiments, the size distribution of theplasma DNA can also be reflected by the fraction of the total DNA lengthcontributed by short DNA fragments. For example, the size distributioncan include a total length of fragments below a cutoff value, which maybe divided by a total length of all fragments, or at least fragmentsbelow a larger cutoff value. Conversely, the size distribution of theplasma DNA can also be reflected by the fraction of the total DNA lengthcontributed by long DNA fragments. For example, the size distributioncan include a total length of fragments above a cutoff value, which maybe divided by a total length of all fragments, or at least fragmentsbelow a smaller cutoff value. As another example, a small vs. largeratio can also be used. One embodiment uses 150 bp as a cutoff to defineshort plasma DNA fragments. However, any cutoff, e.g. 130 bp, 140 bp,160 bp and 170 bp, can also be used as cutoffs to define short DNAfragments. Note that as used herein, base pairs can also refersynonymously with a number of nucleotides (nt) in referring to a lengthof a single stranded fragment.

In one embodiment, a calculation of the fraction of DNA lengthcontributed by short DNA fragments can be as follows: F=Fraction of DNAlength contributed by short DNA fragments; S=Sum of the length of allshort DNA fragments (length equal or below the cutoff); and T=Totallength of all DNA fragments in the sample regardless of their length.The fraction can thus be provided by F=S/T, which is one example of astatistical value of size. A calculation of F may be for all fragmentsfrom a sample or for a particular location of the genome, e.g., for aparticular chromosome.

In one implementation, the total length of all the DNA fragments in asample can be determined. Then, a cutoff size (w) below which the DNAfragments are defined as “short fragments” can be selected. The cutoffsize can be varied and be chosen to fit different diagnostic purposes.The total length of the short DNA fragments can be calculated by summingup the length of all DNA fragments that are equal to or shorter than thecutoff size. The fraction of total length contributed by short DNAfragments can be calculated as follows:F=Σ ^(w)length/Σ⁶⁰⁰length, whereΣ^(w) length represents sum of the lengths of DNA fragments with lengthequal to or less than cutoff w (bp); andΣ⁶⁰⁰length represents the sum of the length of DNA fragments equal to orless than 600 bp. The statistical value F can also be used inembodiments using ranking For example, F can be calculated for fragmentsfrom each of a set of respective genomic locations (e.g. chromosomes).

For illustrative purpose, a total length was calculated by the summationof fragments shorter than 600 bp in the example below. However, othersize limits, e.g. 400 bp, 500 bp and 700 bp, can be used for calculatingthe “total length”. In this example, the total length was calculatedbased on DNA fragments of 600 bp or below because the Illumina GenomeAnalyzer (Solexa) system is not effective in amplifying and sequencingDNA fragments longer than 600 bp. In addition, limiting the analysis toDNA fragments of shorter than 600 bp can also avoid biases arising fromstructural variations of the genome. In the presence of structuralvariation, for example rearrangements (Kidd J M et al, Nature 2008;453:56-64), the size of the DNA fragment can be overestimated when thesize is estimated bioinformatically by mapping the ends of the DNAfragment to the reference genome. In addition, >99.9% of all the DNAfragments successfully sequenced and mapped to the reference genome areless than 600 bp and, thus, including all fragments equal to and shorterthan 600 bp would provide a representative estimation of the sizedistribution of the DNA fragments in the sample.

As discussed above, a shift to a shorter size distribution for DNAfragments can be observed for a chromosome which the fetus has an extracopy. In one aspect, the measurement of the difference in the fractionsof total length contributed by short DNA fragments between thechromosome at risk for the aneuploidy (target chromosome) andchromosomes not being at risk for aneuploidy (reference chromosomes) canbe a quantitative measurement to determine if the size distributions forDNA fragments derived from these chromosomes are different.

In one embodiment, we define F_((Tar)) and F_((Ref)) as the fractions oftotal length contributed by short DNA fragments for the chromosome atrisk of the aneuploidy and the reference chromosome(s), respectively.The difference in the fractions of total length contributed by short DNAfragments between the target chromosome and the reference chromosome(s)(ΔF_((Tar-Ref))) can be calculated as:ΔF_((Tar-Ref))=F_((Tar))−F_((Ref)). For instance, ΔF₍₂₁₋₁₎ is thedifference between the fractions of total length contributed by shortDNA fragments for chromosome 21 and chromosome 1. The application ofΔF_((Tar-Ref)) in the prenatal diagnosis of fetal chromosomalaneuploidies will be discussed in the following sections. In anotherembodiment, a ratio of F_((Tar))/F_((Ref)) can also be used as aseparation value in a similar manner as ΔF_((Tar-Ref)) is used.

A difference between the F values of the target and the referencechromosomes can be used as a statistical value to determine if the fetusis trisomic for the target chromosome or not. When the fetus is trisomicfor the target chromosome, the trisomic chromosome of the fetus wouldcontribute an additional dosage of short fetal DNA to the maternalplasma, hence, resulting in an apparent shortening of the sizedistribution of the target chromosome sequences. This shortening of thesize distribution of the target chromosome sequences would lead to anincrease in the fraction of sequence length contributed by the short DNAfragments for the target sequence (F_(target)). As a result, there wouldbe an increase in the difference ΔF between F_(target) and F_(ref).

FIG. 7 shows a plot 700 of the fraction of total length contributed byshort fragments (F) against the cutoff size (w) for a maternal plasmasample with a male fetus. The F values for the DNA fragments aligned tothe autosomes and chromosome Y are plotted on the vertical axis againstthe cutoff size used for defining “short DNA fragments”. In malepregnancies, the DNA molecules aligned to chromosome Y represent the DNAreleased from the male fetus. As most of the circulating DNA moleculesin maternal plasma are derived from the mother, the DNA fragmentsaligned to the autosomes should represent predominantly the maternal DNAfragments. The F value increases with the cutoff size and approaches thevalue of 1.0 when all DNA fragments in the sample are shorter than orequal to the cutoff size. The difference in size distribution betweentwo species of DNA molecules can be reflected by the difference in theirF values. A higher F value indicates that a higher fraction of the totallength is contributed by the short fragments and, thus, indicates ashorter size distribution of the DNA fragments.

As shown in plot 700, the size distribution for DNA molecules fromchromosome Y is shorter than the size distribution of DNA from theautosomes. Specifically, the F value for chromosome Y rises earlier thanthat for the autosomes, resulting in a higher F_(Y) than F_(autosomes)between 80 bp to 350 bp. The difference in the F values betweenchromosome Y and the autosomes (ΔF_((Y-autosomes))) is further plottedagainst the cutoff size and is represented by a dashed line, which ispositive between 80 bp to 350 bp. The maximum value forΔF_((Y-autosomes)) autosomes) is 0.23 occurring at around 150 bp. Asillustrated in the following examples, the difference in the fraction ofthe total fragment length contributed by the short fragments (ΔF)between the at-risk chromosome and the reference chromosome(s) is auseful separation value to quantify the difference in their sizedistributions. Further by way of example, one could determine the ΔFvalue with any size cutoff, e.g., one between 130 bp and 170 bp.

FIG. 8 shows a plot 800 of the fraction of total length contributed byshort fragments (F) for chromosome 21 (solid line) and the referencechromosomes (all autosomes except chromosomes 13, 18 and 21) (dottedline) against the cutoff size for a euploid pregnancy. The difference inthe two F values (ΔF_((21-Ref))=F_((chr21))−F_((Ref))) is represented bythe dashed line. As the size distributions of DNA fragments fromchromosome 21 and the reference chromosomes are similar in a euploidpregnancy, the value of ΔF_((21-Ref)) is close to zero for any cutoffsize.

FIG. 9 shows a plot 900 of the F values for chromosome 21 (solid line)and the reference chromosomes (all autosomes except chromosomes 13, 18and 21) (dotted line) against the cutoff size for a trisomy 21pregnancy. The ΔF_((21-Ref)) is represented by the dashed line. Due tothe additional dosage of chromosome 21 from the fetus, the sizedistribution of DNA fragments in maternal plasma for chromosome 21 isshorter than that for the reference chromosomes. This difference in thesize distributions of DNA fragments is reflected by the positive valueof ΔF_((21-Ref)), which reaches a maximum of 0.016 at approximately 150bp.

FIG. 10 shows a plot 1000 of ΔF_((21-Ref)) between chromosome 21 and thereference chromosomes (all autosomes except chromosomes 13, 18 and 21)against the size cutoff for a euploid and a trisomy 21 pregnancy. Anincreased ΔF_((21-Ref)) is observed in the trisomy 21 case but theΔF_((21-Ref)) is approximately zero at each size cutoff for the euploidcase. As the maximum ΔF_((21-Ref)) is observed at around 150 bp, thedifference at 150 bp can be used as a separation value for determiningif there is any significant shortening of the size distribution of thechromosome 21 sequences. However, any size at which a significantdifference between euploid and trisomic cases arises can be used, forexample, but not limited to 140 bp, 145 bp, 155 bp and 160 bp. In thisexample, the difference in the fraction of the total length ofchromosome 21 and the reference chromosomes observed at 150 bp are 0.016and −0.002 for the trisomy 21 and euploid pregnancies, respectively.

This difference in the shape of the fraction of the total DNA lengthcontributed by short DNA fragments can be used to distinguish between aeuploid and an aneuploid fetus. A difference can be tested in variousways. In one embodiment, the value of ΔF at a particular size cutoff (anexample of a separation value) can be compared to a cutoff value todetermine a categorization (classification) of a sample. In anotherembodiment, a peak value in ΔF can be found and that value can becompared to one or more categorization cutoff values. In variousembodiments, a peak value can be a maximum or minimum value, an averagevalue near a max/min value, or other value associated with or derivedfrom a max/min value). Other statistical values of a separation value(e.g ΔF) for can also be used, such as the width of the peak, or alocation of the particular size cutoff corresponding to the peak.

In one embodiment, an F value (or other statistical value describedherein, such as length of small fragments divided by length of largefragments) obtained from a plurality of fragments for a particulargenomic location or for the entire genome can be used to determine if apathology exists. For example, if the statistical value exceeds a cutoffvalue, then a pathology can be identified as existing since the amountof small fragments is outside of a normal range. This could be done forpatients besides a pregnant female to identify diseases other than thoseof a fetus.

In some embodiments, a physical size fractionation can be performedprior to a size analysis of fragments. In one embodiment, the nucleicacid molecules can be separated into two size fractions (e.g. one largerthan 200 bp, and one less than or equal to 200 bp), and then a sizedistribution of selected chromosomes (e.g. chromosome 21) on each ofthese size fractions can be compared. In the presence of a fetal trisomy(e.g. trisomy 21), the size fraction of smaller molecular size wouldincrease in relative abundance compared with the size fraction of largermolecular size.

In other embodiments, a number of fragments below a length cutoff valuecan be used instead of a size distribution. For example, the number offragments below a length cutoff value can be compared (e.g. a differenceor ratio) for the target chromosome (e.g. chromosome 21) relative to oneor more reference chromosomes. In one embodiment, the number offragments below the length cutoff value is divided by the total numberof fragments to obtain a percentage, and this percentage can be comparedbetween the target chromosome and the one or more reference chromosomesto provide a parameter. The resulting parameter (e.g. difference orratio) can be compared to a cutoff value (e.g. 1%). In one aspect, thelength cutoff may be chosen at a length where the above percentage isthe highest.

V. Examples Using Rank

In addition to trisomy 21, fragment size analysis in maternal plasma canalso be used for the noninvasive prenatal detection of other fetalchromosomal aneuploidies, such as trisomy 13, trisomy 18 and sexchromosome aneuploidies (such as Turner syndrome, Klinefelter syndromeand XYY, etc). Embodiments can also be used when the chromosomalabnormality involves only a portion of a particular chromosome (e.g.trisomy 21 caused by chromosomal translocation). In such a scenario,fragment size abnormalities will be observed for DNA fragments from theaffected chromosomal regions.

FIG. 11 shows a table 1100 for libraries of maternal plasma DNA thatwere constructed using a multiplexing sample preparation kit (Illumina)according to the manufacturer's instructions. Every two samples withdistinguishable barcodes were introduced into one lane and subsequentlysubjected to standard multiplexed paired-end sequencing on an IlluminaGenome Analyzer II. The samples could be distinguished on the basis ofbarcodes. The plasma samples of 120 pregnant women were analyzed. Thesex and the chromosomal aneuploidy status of the fetuses are shown inTable 1100.

FIG. 12 illustrates the ranking of the different chromosomes for foursamples of different disease or non-disease states according toembodiments of the present invention. The 22 autosomes, together withchromosome X, were ranked according to the size of their fragments asdescribed earlier. As can be seen, the relative ranking of thechromosomes based on version 2 of the Illumina Cluster GenerationReagent Kit is different from when version 1 of the kit was used (FIG.2). As shown below, embodiments (e.g. method 400) allow an aneuploidcase to be differentiated from a euploid case.

A. Trisomy 13

In this example, we demonstrate the use of embodiments for the prenataldiagnosis of trisomy 13. FIG. 13 shows a graph of the rank of chromosome13 for the 120 pregnancies, e.g., as may result from method 400. In thegraph, T13, T18, T21 and Eu refer to trisomy 13, trisomy 18, trisomy 21and euploid pregnancies, respectively. In 18 (78.3%) of the 23 trisomy13 pregnancies, chromosome 13 ranked 22 or below whereas in only 2(2.1%) of the 97 non-trisomy 13 pregnancies, chromosome 13 ranked 22 orbelow. Therefore, using a cutoff ranking of 22, the sensitivity andspecificity of rank analysis of chromosome fragment size for prenataldiagnosis of fetal trisomy 13 were 78.3% and 97.9%, respectively.

In FIG. 13, one can see that the rank of chromosome 13 was higher (i.e.with a smaller number denoting the rank) for euploid and trisomy 18 and21 pregnancies than for trisomy 13 pregnancies. In other words, whencompared with sequences on the other chromosomes, the chromosome 13sequences appear to be shorter for the trisomy 13 pregnancies than forthe non-trisomy 13 pregnancies. The apparent shortening of chromosome 13sequences in trisomy 13 pregnancies is due to the increased contributionof fetal DNA to chromosome 13 due to the extra chromosome 13 of thefetus.

B. Trisomy 18

FIG. 14 shows a ranking of chromosome 18 for the 120 cases. In 26(86.7%) of the 30 trisomy 18 cases, the ranks of chromosome 18 werelower (i.e. with a large number denoting the rank) than 13 whereas noneof the 90 non-trisomy 18 cases had a rank lower than 13. Therefore,using a rank of 13 as a cutoff, the sensitivity and specificity ofranking analysis of chromosome fragment size for the prenatal diagnosisof fetal trisomy 18 are 86.7% and 100%, respectively.

In this analysis, we have compared the size ranking of chromosome 18 intrisomy 18, trisomy 13, trisomy 21, and euploid pregnancies. In thecontext of chromosome 18, the latter three groups can both be regardedas ‘normal’ controls because they do not have dosage abnormalitiesinvolving chromosome 18. As can be seen from FIG. 14, the rank forchromosome 18 for euploid and trisomy 13 cases are clustered around 1 to3. On the other hand, the ranks for chromosome 18 for the trisomy 18cases are 13 to 22, indicating that the size of chromosome 18 fragmentsis shorter than that in the euploid, trisomy 21, and trisomy 13 cases.Again, the extra chromosome 18 explains these observations.

C. Trisomy 21

FIG. 15 shows a ranking of chromosome 21 for the 120 cases. In 8 (88.9%)of the 9 trisomy 21 cases, chromosome 21 ranked 22 or lower whereas innone of the 111 non-trisomy 21 cases, chromosome 21 had a rank of 22 orlower. Therefore, using a rank of 22 as a cutoff, the sensitivity andspecificity of ranking analysis of chromosome fragment size for theprenatal diagnosis of fetal trisomy 21 would be 88.9% and 100%,respectively.

V. Examples Using Difference In Size

A. Trisomy 13

In these next examples, we demonstrate that one can achieve thenoninvasive prenatal detection of trisomy 13 by comparing the absolutesizes of fragments derived from chromosome 13, with those of fragmentsderived from one or more reference chromosomes, e.g. as described formethod 600. This example utilizes the same dataset as that in theprevious example. As an illustration, for trisomy 13 detection, we haveselected chromosome 5 and chromosome 6 as reference chromosomes.

As can be seen from table 1600 of FIG. 16, within the same sample, thesequences aligned to chromosome 13 were significantly shorter than thesequences aligned to chromosome 5 and chromosome 6 for all the trisomy13 pregnancies (Mann-Whitney rank-sum test, p-value≦0.001). As controls,euploid and trisomy 18 pregnancies were included. In both euploid andtrisomy 18 pregnancies, the dosage of chromosome 13 was normal. As canbe seen from table 1600, size abnormalities of such statisticalsignificance for chromosome 13-derived sequences were not seen ineuploid and trisomy 18 pregnancies.

Furthermore, for all the trisomy 13 pregnancies, within the same sample,the difference of the mean fragment size between chromosome 13 andchromosome 5 was greater than 0.4 bp whereas none of the non-trisomy 13cases showed a difference more than 0.4 bp. Similarly, for all thetrisomy 13 pregnancies, the difference of the mean fragment size betweenchromosome 13 and chromosome 6 was greater than 0.5 bp whereas none ofthe non-trisomy 13 cases showed a difference more than 0.5 bp.

B. Trisomy 18

As an illustration, for trisomy 18 detection, chromosome 14 has beenselected as the reference chromosome. For non-trisomy 18 pregnancies, itcan be seen in table 1700 of FIG. 17 that sequences derived fromchromosome 18 are statistically significantly longer than those derivedfrom chromosome 14 (Mann-Whitney rank-sum test, p-value≦0.005). However,for the trisomy 18 cases, the chromosome 18 sequences were notsignificantly longer than sequences aligned to chromosome 14. Theeuploid and trisomy 18 cases can be differentiated based on thedifference between the mean size of DNA fragment from chromosomes 18 and14 using a cutoff value of 0 bp. These observations can be explained bythe fact that the additional dose of fetal-derived chromosome 18sequences which are shorter than maternal ones would reduce the overallsize of such sequences when the fetus has trisomy 18. This would thenbring the overall size distribution of chromosome 18 closer to thedistribution of chromosome 14.

V. Examples Using Total Length Contribution

In the examples below, the reference chromosomes consisted of all theautosomes except chromosomes 13, 18 and 21.

FIG. 18 shows a difference in the fractions of the total lengthcontributed by short fragments between chromosome 18 and the referencechromosomes (ΔF_((18-Ref))) at 150 bp.

Using a diagnostic cutoff value of 0.0003 for ΔF_((18-Ref)), the trisomy18 pregnancies can be detected with 93.3% sensitivity and 100%specificity.

FIG. 19 shows a difference in the fractions of the total lengthcontributed by short fragments between chromosome 21 and the referencechromosomes (ΔF_((21-Ref))) at 150 bp. Using a diagnostic cutoff valueof 0.007 for ΔF_((21-Ref))), the trisomy 21 pregnancies can be detectedwith 100% sensitivity and 100% specificity.

VII. Selection of Reference Chromosome(S)

One or more reference chromosomes can be selected in various ways forthe noninvasive prenatal detection of fetal chromosome aneuploidy bysize analysis of maternal plasma DNA, e.g. using method 600. In variousembodiments, different reference chromosomes can be selected.

A first type of reference chromosomes are those in which DNA fragmentsderived from them exhibit a similar size distribution on a specificanalytical platform (or analytical platforms with closely matchedanalytical performances) as those derived from the chromosomepotentially involved in an aneuploidy in maternal plasma (e.g.chromosome 21, 18, or 13). In one embodiment, in this type of analysis,a fetal chromosomal aneuploidy is detected if the size ranking orabsolute size of the at-risk chromosome shows a statisticallysignificant reduction from the reference chromosome(s). In otherembodiments, one can measure the mean or median size difference betweenfragments derived from the at-risk chromosome and those from thereference chromosome(s).

A second type of reference chromosomes are those in which DNA fragmentsderived from them are statistically shorter than those derived from thechromosome potentially involved in an aneuploidy in maternal plasma(e.g. chromosome 21, 18, or 13) when the fetus is euploid. This type ofscenarios may be encountered when the at-risk chromosome is one of thelongest when measured using a particular platform. For example in FIG.12, chromosome 18-derived DNA fragments were the longest amongst theautosomes when measured in maternal plasma. Thus, one can selectreference chromosomes whose fragments are statistically significantlyshorter than chromosome 18. In one embodiment, in this type of analysis,a fetal chromosomal aneuploidy is detected in a case if the size rankingor absolute size of the at-risk chromosome cannot be seen to bestatistically significantly different from the reference chromosome(s).For example, this strategy has been used for the analysis for FIG. 17,as described above.

A third type of reference chromosomes are those in which DNA fragmentsderived from them are statistically longer than those derived from thechromosome potentially involved in an aneuploidy in maternal plasma(e.g. chromosome 21, 18, or 13) when the fetus is euploid. This type ofscenarios may be encountered when the at-risk chromosome is one of theshortest when measured using a particular platform. Thus, one can selectreference chromosomes whose fragments are statistically significantlylonger than the at-risk chromosome. In one embodiment, in this type ofanalysis, a fetal chromosomal aneuploidy is detected in a case if thesize ranking, difference in ranking or absolute size between thereference and the at-risk chromosome is increased.

A fourth type of reference chromosomes are those with similar GCcontent. The GC content of a chromosome can affect the quantitativereadout in a sequencing reaction. One way to minimize potential biasresulting from a difference in GC content between chromosomes is toselect appropriate reference chromosomes with similar GC content. FIG.20 shows a list of the GC contents of different chromosomes (NCBI build36, version 48). The chromosomes are listed in ascending order of the GCcontent. An example of using reference chromosomes with similar GCcontent is now provided.

FIG. 21 shows a difference in the fractions of the total lengthcontributed by short fragments between chromosome 13 and the referencechromosomes (ΔF_((13-Ref))) at 150 bp. Here, by way of illustration, weused chromosomes 3, 4, 5 and 6 as the reference chromosomes for theΔF_((13-Ref)) analysis. As shown in FIG. 20, chromosomes 3-6 have GCcontent of 36.53%-38.79%, which is similar to GC content of 39.07% forchromosome 13. Using a diagnostic cutoff of 0.0038, the ΔF analysisdetected trisomy 13 cases with a sensitivity of 95.7% and a specificityof 99.0%.

It is expected that the GC bias issue may affect different sequencingplatforms to different extents. For example, the use of platforms whichdo not require prior amplification, such as the Helicos platform (HarrisT D, et al. Single-molecule DNA sequencing of a viral genome. Science2008; 320:106-9), nanopore (Lund J, Parviz B A. Scanning probe andnanopore DNA sequencing: core techniques and possibilities. Methods MolBiol 2009; 578:113-22), or the single molecule real time system fromPacific Biosciences (Eid J, et al. Real-time DNA sequencing from singlepolymerase molecules. Science 2009; 323:133-8), might allow a broaderchoice of the reference group of chromosomes.

IV. Use of Fetal DNA Concentration

In trisomy 21 (or other trisomy) pregnancies, the fetus will release anextra dose of chromosome 21 fragments, which would be shorter than thosefrom maternal cells, into maternal plasma. One would expect that theconcentration of these shorter fragments will be correlated with theconcentration of fetal DNA in maternal plasma. In other words, thehigher the fractional concentration of Y chromosome-derived sequences,the shorter will be the measured size of chromosome 21-derived sequencesin trisomy 21 pregnancies.

FIG. 22 illustrates the results from a number of pregnancies involvingmale trisomy 21 fetuses. As can be seen in FIG. 22, there is indeed anegative correlation between the median size of chromosome 21 sequencesand the percentage of sequences aligned to the Y chromosome (r=−0.942,Pearson correlation). A similar trend would also be expected if the sizeranking of chromosome 21 is used, i.e., the rank number of chromosome 21will increase, indicating shorter fragments when the fractional fetalDNA concentration is increased. As there is a correlation, embodimentscan use a measurement of fetal DNA concentration as a parameter in anyof the methods described herein.

In this type of analysis, the fractional and absolute concentration offetal DNA in maternal plasma can be measured by any method known tothose skilled in the art. If the fetus is male, the concentration offetal DNA can be measured by the fractional concentration of sequencesderived from the Y chromosome in maternal plasma. Another example is theuse of paternally-inherited genetic markers such as single nucleotidepolymorphisms or simple tandem repeat polymorphisms orinsertion-deletion polymorphisms. Another example is the use ofepigenetic markers such as regions that are differentially methylatedbetween fetal and maternal DNA (Poon et al. Clin Chem 2002; 48: 35-41;Chiu et al. Am J Pathol 2007; 170: 941-950; Chan et al. Clin Chem 2006:52: 2211-2218; U.S. Pat. No. 6,927,028). The above markers can beanalyzed using methods known to those of skill in the art, includingpolymerase chain reaction (PCR), digital PCR, sequencing, massivelyparallel sequencing and targeted massively parallel sequencing.

In one embodiment, one could vary the diagnostic threshold (e.g. cutoffvalue for any method described herein) for detecting a chromosomalaneuploidy in relation to the measured fetal DNA concentration inmaternal plasma. Thus, for maternal plasma samples with a relativelyhigh fetal DNA, the degree of shortening of plasma DNA molecules derivedfrom the chromosome potentially involved in the aneuploidy would beexpected to be more dramatic than for maternal plasma samples with arelatively low fetal DNA concentration.

As ΔF is related to the size distribution, ΔF also shows a correlationwith fetal DNA concentration. FIG. 23A shows a correlation betweenΔF_((18-Ref)) (chromosome 18 vs. reference chromosomes) and fetal DNAconcentration. Ten of the thirty T18 cases were carrying male fetuses,and thus the fractional concentration of fetal DNA can be estimated bythe fractional concentration of chromosome Y sequences in these samples.There was a significant correlation between ΔF_((18-Ref)) and thefractional concentration of chromosome Y sequences (r=0.879, Spearmancorrelation). These results suggest that the degree of shortening in thesize distribution of chromosome 18 sequences in maternal plasma iscorrelated with the fractional concentration of fetal DNA in thematernal plasma in trisomy 18 pregnancies.

The cases below the diagnostic cutoff are represented by an open circlewhereas the cases having a difference of more than the cutoff arerepresented by filled circles. The two cases which had ΔF values lessthan the diagnostic cutoff (0.0003) (represented by open circles) hadrelatively lower fractional concentrations of fetal DNA when comparedwith those with ΔF values greater than 0.0003 (represented by filledcircles). The low fractional fetal DNA concentrations might be thereason why these two cases were missed in the analysis performed in FIG.18. Accordingly, in one embodiment, if fetal concentration is low in asample, the classification might be disregarded or redone.

FIG. 23B shows a correlation between ΔF_((21-Ref)) (chromosome 21 vs.reference chromosomes) and fetal DNA concentration. Five of the nine T21cases were carrying male fetuses. There was a significant correlationbetween the value of ΔF_((21-Ref)) and the fractional concentration ofchromosome Y sequences (r=0.9, Spearman correlation).

FIG. 23C shows a correlation between ΔF_((13-Ref)) (chromosome 13 vs.chromosomes 3, 4, 5 and 6) and fetal DNA concentration. Fourteen of thetwenty-three trisomy 13 cases were carrying male fetuses. The fractionalconcentration of fetal DNA can be estimated by the fractionalconcentration of chromosome Y sequences in the samples. There was apositive correlation between ΔF_((13-Ref)) and fractional concentrationof chromosome Y sequences (r=0.644, Spearman correlation). The casewhich has a ΔF_((13-Ref)) below the diagnostic cutoff (0.0038) isrepresented by an open circle. The low fractional fetal DNAconcentrations might be the reason why this case had been missed in theanalysis performed in FIG. 21.

IX Comparison of Size Analysis and a Molecular Counting Method

FIG. 24 shows a comparison of the accuracies of an embodiment of thepresent invention and another method for the noninvasive detection offetal aneuploidies (trisomy 13 and trisomy 18) using maternal plasma DNAanalysis. This example illustrates a comparison of embodiments usingsize versus a method based on molecular counting (U.S. patentapplication Ser. No. 11/701,686; Chiu et al Trends Genet 2009; 25:324-331; Chiu et al Proc Natl Acad Sci USA 2008; 105: 20458-20463; Fanet al Proc Natl Acad Sci USA 2008; 105: 16266-16271; US PatentPublication 2009/0029377). Eight maternal plasma samples (two euploid,two trisomy 18, and four trisomy 13) were analyzed using the methoddescribed by Chiu et al (Proc Natl Acad Sci USA 2008; 105: 20458-20463).For each case, the previously reported molecular counting method usingz-score by Chiu et al with molecular counting was compared with theresults of embodiments based on size analysis.

For the calculation of the z-score, the percentage representation of thechromosome of interest was first calculated for each case. Then, themean and standard deviation of chromosomal representation was calculatedfor the reference cases. In this data set, cases 1, 2, 5, 6, 7 and 8were used as the reference group for a calculation of the mean andstandard deviation of the chromosome 18 representation. Cases 1, 2, 3and 4 were used as the reference group for a calculation of the mean andstandard deviation of the chromosome 13 representation. The z-score isdefined as the number of standard deviations from the mean of thereference group. Significant overrepresentation of a chromosome isdefined as a z-score of >3. Cases 3 and 4 were carrying trisomy 18fetuses and the chromosome 18 fragments were overrepresented in theirplasma. Cases 5, 6, 7 and 8 were carrying trisomy 13 cases, but onlycases 5 and 7 showed an overrepresentation of chromosome 13 in theirplasma. Cases 6 and 8 did not show significant overrepresentation ofchromosome 13 in maternal plasma despite carrying trisomy 13 fetuses.

Significant shortening of the DNA fragments size of a chromosome wasdetected by comparing the size of all the fragments aligned to aparticular chromosome to those aligned to a reference chromosome. TheMann-Whitney test was used for the comparison, and a P-value of <0.0001was defined as the presence of a significant difference. For theanalysis of the size of chromosome 13 fragments, the referencechromosome was chromosome 5. For all the cases carrying a euploid ortrisomy 18 fetus, the fragment size of chromosome 13 was notsignificantly different from that of chromosome 5. For the four casescarrying trisomy 13 fetuses, the chromosome 13 fragments weresignificantly shorter than chromosome 5 fragments, implying that thefragment size of chromosome 13 was shortened compared with thenon-trisomy 13 cases. Thus, all four trisomy 13 cases were correctlyidentified by the present invention, compared with two out of four bythe z-score method.

For the analysis of the size of chromosome 18 fragments, the referencechromosome was chromosome 12. For all the cases carrying a euploid ortrisomy 13 fetus, the fragment of chromosome 18 was significantly longerthan the chromosome 12 fragments. For the two cases carrying trisomy 18fetuses, the sizes of chromosome 18 fragments were not significantlydifferent from those of chromosome 12 fragments, implying that thefragment size of chromosome 18 was shortened when compared with casescarrying non-trisomy 18 fetuses. In other words, both trisomy 18 caseswere correctly classified.

X. Detection of Polymorphisms and Diagnosis of Genetic Disorders

Size analysis of maternal plasma DNA can also be used for thenoninvasive detection of fetal genotype. The fetal genotype can be usedto determine whether the fetus has inherited a mutated gene, has animbalance of a particular allele, or other sequence imbalances orpurposes. In such embodiments, one allele can be a reference genomiclocation (sequence) and a different allele can be the genomic locationunder test. Thus, any of the methods using a reference sequence can alsobe applied to determining a genotype and other sequence imbalances.

In one embodiment, a sequence imbalance (and thus possibly a genotype)can be determined by whether a size difference (imbalance) does or doesnot exist between alleles in the maternal sample (e.g. when the motheris heterozygous at the allele). For example, if there is no differencein the size profile between alleles in the sample, then the fetus may bedetermined to have the same genotype as the mother. As another example,if there is a difference in the size profile between alleles in thesample, then the fetus may be determined to have a genotype differentfrom that of the mother.

In the following examples, the mother is heterozygous (i.e., with onecopy of the N allele and one copy of the M allele, denoted by NM) for aparticular locus. The letters N and M nominally represent the wildtype(N for normal) and mutant (M for mutant) alleles, respectively. However,the N and M alleles can correspond to any two different alleles, and notnecessarily wildtype and/or mutant. In one embodiment, M can beconsidered the at-risk genomic sequence and N the reference sequence.With this context, one can understand the application of any of theabove methods using a reference sequence to the determination of agenotype.

In a non-pregnant woman, the average size of molecules carrying both ofthe two alleles would be the same. However, in the plasma of a pregnantwoman, there is a mixture of DNA molecules from the mother and thefetus. The maternal-derived DNA molecules are longer than thefetal-derived ones. If the mother and fetus both have two alleles (i.e.both the N and M alleles), both of these alleles will have an equalcontribution of long and short DNA molecules. Thus, the resulting sizedistribution of the N and M alleles will be the same. Conversely, if thegenotypes of the mother and fetus are different, e.g. if the mother isNM and the fetus is MM, then the size distribution of the N and Malleles will be different. In other words, the size distribution of thetwo alleles in plasma would be affected by the genotype of the fetus.FIGS. 25A-25C show diagrams for different scenarios for genotypes of apregnant woman and the fetus according to embodiments of the presentinvention.

In FIG. 25A, the fetus has a genotype of NN, and the genotype of themother is NM. The length of a bar 2510 is an indication of an average(mean) size of fragments that are from one of the two alleles of themother and of the fetus, respectively. As described above, a fetus has asmaller average size than a mother. Thus, the long bars representmaternal DNA and the short bars represent fetal DNA.

Since both the mother and the fetus contribute to the allele N whereasonly the mother contributes to the allele M, the size distribution formolecules with the allele N would be shorter than that for moleculeswith the allele M. In other words, in a pregnant woman who has thegenotype of NM, a shorter size distribution for allele N compared withallele M would imply the fetal genotype being NN. Thus, it can beidentified that the fetus has two wildtype (N) alleles when the sizedistribution (e.g. mean size) of N is smaller than M by a certain cutoff(e.g. a percentage or absolute value).

In FIG. 25B, the fetus has the genotype of NM. Both the mother and fetuscontribute alleles N and M. As a result, the size distributions formolecules with the alleles M and N are the same. In a pregnant woman whohas the genotype of NM, the same size distributions for alleles M and Nwould indicate a fetal genotype of NM. Thus, it can be identified thatthe fetus has one wildtype (N) allele and one mutant (M) allele when thesize distribution (e.g. mean size) of N is about equal to M within acertain cutoff (e.g. percentage or value).

In FIG. 25C, the fetus has the genotype of MM. Since both the mother andthe fetus contribute to the allele M whereas only the mother contributesto the allele N, the size distribution for molecules with the allele Mwould be shorter than that for molecules with the allele N. In apregnant woman who has the genotype of NM, the shorter size distributionfor allele M than allele N would indicate the fetal genotype being MM.Thus, it can be identified that the fetus has two mutant (M) allele whenthe size distribution (e.g. mean size) of M is smaller than N by acertain cutoff (e.g. percentage or value).

The method can also be used to analyze situations where the mother ishomozygous, e.g., NN or MM. If the fetus has a different genotype, thesize distribution from the maternal sample will also change, and thusthe genotype of the fetus can be determined. Also, if the sizedistribution does not change, then the genotype may be determined to bethe same as the mother's, as described above for the case where themother is heterozygous case.

In some embodiments, the determination of whether there is an imbalance,or otherwise, relative to the mother's genotype (e.g. one N for one Mwould imply no imbalance) can be performed with cutoff values. Forexample, if there is a large enough deviation (e.g. by percentage) fromthe mother's genotype, then the fetus can be determined to haveinherited the allele with the smaller size distribution. In oneembodiment, the cutoff value can be dependent on a percentage of fetalnucleic acids in the maternal sample. If there is a higher percentage offetal nucleic acids, then a larger deviation would be expected, and thusa larger cutoff can be used (e.g. for the difference in sizedistribution for one allele relative to another). If there is a lowerpercentage of fetal nucleic acids, then a smaller deviation would beexpected, and thus a smaller cutoff may be used.

In one embodiment, a genotype of the father may be used to determinewhich allele of the mother might have its size distribution changed dueto the fetal nucleic acids. In instances where the father is homozygousfor the genotype, this can allow for narrowing the possible fetalgenotype to just what allele is inherited from the mother, since whichone came from the father is known. After such a narrowing, thedetermination of the fetal genome might be more accurate since only twopossibilities would need to be tested. In one implementation, a samecutoff value is used regardless of which genotype is inherited from thefather. In another implementation, a different cutoff can be used.

In various embodiments, any of the size distributions mentioned hereinmay be used in such sequence imbalance determinations. In someembodiments, an accuracy level may also be provided For example, aclassification of “undetermined” may also be used, besides an imbalanceand a balance classification. In this manner, some determinations may bedetermined with high confidence, whereas values in a middle region mayrequire further data points.

XI. Size Analysis for Fetal Haplotype Inheritance

The application of size analysis can further be extended to determinewhich maternal haplotype is passed onto the fetus. A haplotype can referto alleles at multiple loci. A definition of the term “haplotype” can befound in the Definitions section of the present application. The fetalhaplotype can be used to determine whether the fetus has inherited amutated gene, has an imbalance of a particular allele, or otherpurposes. Thus, the haplotype may be used in a similar manner as thegenotype, but since there are more loci, a smaller volume of bloodsample may be used to achieve the same, or even better, statisticalconfidence of determining the fetal haplotypes. In one aspect, thesequence imbalance can be determined relative to the mother's haplotype.Herein, a haplotype can be represented as a series of polymorphisms, forexample SNPs, each at a particular location in the genome, at which asequence variation is known.

In one embodiment, a method for determining a haplotype of a fetus byanalyzing for sequence imbalances in the maternal plasma is provided. Inone aspect, separation values (e.g. differences) in the size profilesbetween haplotypes is used to determine the sequence imbalance. In oneembodiment, the haplotypes of the mother (e.g., Hap I and Hap II) aredetermined by analysis (e.g. sequencing (He D et al. Bioinformatics2010; 26: i183-i190) or single molecule haplotyping (Ding C et al. ProcNatl Acad Sci USA 2003; 100: 7449-7453 and Xiao M et al. Nat Methods2009; 6: 199-201) of a maternal sample (e.g. a sample that does notcontain fetal nucleic acids). In another embodiment, the haplotype(s) ofthe mother can be determined using an analysis of the mother's parents,siblings, a previous child, or other relatives. In yet anotherembodiment, for polymorphisms at strong linkage disequilibrium, knowingthe mother's genotype at one locus can imply a genotype at other loci,e.g., when alleles normally appear in a same sequence, i.e. haplotype.Thus, the mother's haplotype can implicitly be determined from onemeasure of genotype. The genotype at more than one locus can also bedetermined, where each determined genotype can imply genotypes at otherloci and hence the haplotype is deduced.

In one embodiment, the father's genotype is also determined. Thisinformation can be used to determine if the father is homozygous orheterozygous at particular SNPs. A direct determination of the father'sallele at each locus can be made. Accordingly, one can determine if eachpaternal allele is the same as the allele on Hap I or Hap II of themother, termed type α or type β SNP.

In another embodiment, the father's haplotype is also determined. Thefather's haplotype can be determined by analysis (e.g. sequencing (He Det al. Bioinformatics 2010; 26: i183-i190) or single moleculehaplotyping (Ding C et al. Proc Natl Acad Sci USA 2003; 100: 7449-7453and Xiao M et al. Nat Methods 2009; 6: 199-201) of a paternal sample, orthrough an analysis of the father's parents, siblings, a previous child,or other relatives. Alternatively, for polymorphisms at strong linkagedisequilibrium, knowing the father's genotype at one locus can imply agenotype at other loci, e.g., when alleles normally appear in a samesequence, i.e. haplotype. Thus, the father's haplotype can implicitly bedetermined from one measure of genotype. The genotype at more than onelocus can also be determined, where each determined genotype can implygenotypes at other loci and hence the haplotype is deduced.

One application of such embodiments can be to determine if a sequenceimbalance (e.g. by detecting a difference in size profile) does or doesnot exist in a maternal sample containing fetal nucleic acids byanalyzing SNPs where both the father and mother are heterozygous. Thefetus's genotype or haplotype is thereby deduced from whether thesequence imbalance does or does not exist.

FIG. 26 shows an example where the mother is heterozygous and the fatheris homozygous at the SNP loci to be analyzed. To carry out the sizeanalysis, the focus can be on a subset of SNPs which the mother isheterozygous and the father is homozygous. The two homologouschromosomes of the mother are termed Hap I and Hap II, respectively. Foreach of these SNPs, we can determine which one of the two maternalalleles is located on Hap I and which is on Hap II. A SNP is defined astype α if the paternal alleles are identical to the maternal allele onHap I, and as type β if the paternal alleles are identical to thematernal allele on Hap II. A further description of determinations of afetal genome can be found in the application “Fetal Genomic AnalysisFrom A Maternal Biological Sample” referred to above.

Once the mother's haplotype and father's genotypes or haplotype areknown, a size distribution for fragments associated with each SNP can beanalyzed to determine the fetus's haplotype by identifying whether thereis a sequence imbalance among the SNP subset. In an alternativeembodiment, the paternal genotype or haplotype is not known for certain,one could nonetheless deduce using, for example, a statistical procedurethe likely paternal genotype or haplotype, based on, for example, thefrequencies of known genotypes or haplotypes in the tested population. Asequence imbalance (as determined via a size imbalance) is thendetermined for each SNP, e.g., as discussed in section X.

For example, if the father is homozygous for an allele on Hap I (typeα), then either there will be no size imbalance (fetus inherits Hap IIfrom mother and thus is heterozygous for Hap I and Hap II just like themother) or there will be a size imbalance where Hap I has a smaller sizedistribution (fetus inherits Hap I from mother and thus is homozygousfor Hap I). If the father is homozygous for Hap II (type β as describedbelow), then either there will be no imbalance (fetus inherits Hap Ifrom mother and thus is heterozygous for Hap I and Hap II) or there willbe an imbalance where Hap II has a smaller size distribution (fetusinherits Hap II from mother and thus is homozygous for Hap I). Anindeterminate classification can also be used, or varying levels ofcertainty between a balance and an imbalance. Typically, a similarcutoff would be used for either type. In various embodiments, any of thesize distributions mentioned herein may be used.

In one embodiment, a size distribution for the fragments for each of aplurality of SNP location can be analyzed to determine the twohaplotypes of the fetus. For example, a difference between the sizedistribution of fragments for the SNP alleles on one haplotype can becompared to the size distribution of SNP alleles on the other haplotype.The statistics can be analyzed in various ways, for example, by making adetermination for each SNP and then taking the majority (balance,imbalance, and maybe including indeterminate) as the haplotype. Asanother example, the sizes can be aggregated across SNPs (e.g. to obtainan average or median size distribution that is compared to a cutoffvalue). Or, a combination of the two can be used. Another implementationcould be to use an extremum of the data points, e.g., the smallestdifference for a particular SNP.

FIG. 27 shows an example where the fetus inherits Hap I from the motherwhen the paternal haplotype is as shown in FIG. 26. For type α SNPs(enclosed by non-shaded boxes), the fetus would have inherited allelesidentical to the ones located on maternal Hap I from the father. Hence,the fetus would be homozygous for the alleles on Hap I. As a result thesize distribution of the alleles on Hap I would be shorter than theallele on Hap II in maternal plasma. For type β SNPs (enclosed by shadedboxes), the fetus would have inherited alleles identical to those on thematernal Hap II from the father. Hence, the fetus would be heterozygous.As a result, the size distribution of the alleles on Hap I and Hap IIwould have the same size distribution in maternal plasma.

In one embodiment, SNPs of the same type (type α or type β) can beanalyzed together. For type α SNPs, the size distribution of moleculescarrying the alleles on Hap I would be shorter than the sizedistribution of molecules carrying the alleles on Hap II. For type βSNPs, the size distributions of molecules carrying the alleles on Hap Iand Hap II would be the same. In other words, if the size distributionfor molecules carrying Hap I is shorter than that for molecules carryingHap II, the fetus is homozygous for Hap I. If the size distributions formolecules carrying Hap I and II are the same, the fetus is heterozygous.

EXAMPLE

The following experiment was used to test the accuracy of a fetalhaplotype inheritance analysis. A couple, attending an obstetrics clinicfor the prenatal diagnosis of β-thalassemia, was recruited. Bloodsamples were taken from the father and mother. For the mother, the bloodsample was taken prior to chorionic villus sampling (CVS) at 12 weeks ofgestation. Following CVS, a portion was stored for the experiment.

DNA was extracted from the buffy coats of the father and mother, and theCVS sample. These DNA samples were subjected to analysis by theAffymetrix Genome-Wide Human SNP Array 6.0 system to determine thegenotypes of the father, mother and the fetus. In this experiment, theCVS data were used to deduce the maternal haplotype. However, in theclinical implementation of the test, the maternal haplotype could bededuced by other means as described above. The CVS data were also usedto confirm the accuracy of the determination using methods herein, whichdo not require a CVS.

In the current illustration, we focus on an informative subset of SNPsin which the mother was heterozygous and the father was homozygous. Inthis subset of SNPs, the genotypes of the couple and the fetus were usedfor constructing the haplotypes of the mother. We defined that haplotypeI (Hap I) to be the series of alleles that the mother had passed ontothe fetus whereas haplotype II (Hap II) to be the series of alleles thatthe fetus did not receive from the mother.

Then, we divided the informative SNPs into two subtypes, namely type αand type β. For type α SNPs, paternal alleles were identical to thematernal allele on Hap I. For these SNPs, the fetus would have inheritedthe same allele (the allele on Hap I) from the parents and, hence, thefetus would be homozygous for the SNPs on Hap I. For type SNPs, thepaternal alleles were identical to the maternal allele on Hap II. Forthese SNPs, the fetus would have inherited the alleles on Hap I from themother and different alleles (identical to the ones on Hap II) from thefather and, hence, the fetus would be heterozygous.

DNA extracted from the plasma of the mother was subjected to massivelyparallel sequencing using the Illumina Genome Analyzer platform.Paired-end sequencing of the plasma DNA molecules was performed. Eachmolecule was sequenced at each end for 50 bp, thus totaling 100 bp permolecule. The two ends of each sequence were aligned to thenon-repeat-masked human genome (Hg18 NCBI.36 downloaded from UCSCgenome.ucsc.edu) using the SOAP2 program from the Beijing GenomicsInstitute at Shenzhen (soap.genomics.org.cn/) (Li Ret al. Bioinformatics2009, 25(15):1966-7)

In one embodiment, a statistical value of fraction of total lengthcontributed by short fragments for haplotypes was used to determinewhich maternal haplotype was passed onto the fetus. As an example,chromosome 22 is used to illustrate how the size analysis can be usedfor deducing which maternal haplotype is passed onto the fetus. First,we divided chromosome 22 into several segments with each segmentcontaining 50 informative SNPs in which the mother was heterozygous andthe father was homozygous (chromosome 22 and the segments both beingexamples of sequences). For each segment, the DNA fragments (examples ofmolecules that are part of the sequences) covering these informativeSNPs were divided into two groups, namely Hap I and Hap II, according towhich of the two maternal haplotypes these fragments corresponded to.For each segment, the sum of total length of all fragments mapping tothe maternal Hap I and maternal Hap II were determined. Then, similarly,the sum of total length of short fragments mapping to the maternal Hap Iand Hap II in each segment were determined. For illustration purpose,fragments of 150 bp or shorter are defined as short in this example forthe calculation of the total length of short fragments. From theselengths, the fractions of total length contributed by short fragmentscan be calculated for the DNA fragments mapping to Hap I and Hap IIwithin each segment.

FIG. 28 shows a table illustrating a size analysis for type α SNPs onchromosome 22 according to embodiments of the present invention.ΔF_((Hap I-Hap II)) represents the difference in the fraction of totallength contributed by short fragments between Hap I and Hap II. When thelast segment which consisted of only 28 SNPs is excluded,ΔF_((Hap I-Hap II)) ranged from 0.0288 to 0.0701. The positive values ofΔF_((Hap I-Hap II)) signify that the F values of Hap I are alwaysgreater than that of Hap II. As the F value is defined as the fractionof total length contributed by short fragments, a higher F valueindicates that a higher fraction of total length is contributed by shortfragments. In other words, these results indicate that the DNA fragmentscarrying alleles on Hap I are shorter than those carrying alleles on HapII for each of the regions consisting of 50 SNPs being analyzed. Thisindicates that the size distribution of Hap I is shorter than that ofHap II. Therefore, we can deduce that the fetus is homozygous for thealleles on Hap I. As mentioned above, in one embodiment, aclassification can be made for each segment based on the separationvalue (e.g. ΔF) between the two genomic locations in Hap I and Hap II,and then a total classification can be made based on the respectivesegment classifications. In another embodiment, a total separation value(e.g. an average separation value) can be determined from the separationvalues for each segment, and this total statistical value can be used todetermine a classification.

In one embodiment, a cutoff of about 0.02 may be used to determine if animbalance exists. If a median or average of ΔF was used, a larger cutoffcould be used and still be accurate. Cutoffs may also be used forindeterminate results, e.g., a region between 0.015 and 0.025 may beindeterminate and in need of further analysis.

FIG. 29 shows a table illustrating a size analysis for type β SNPs onchromosome 22 according to embodiments of the present invention. ΔF(HapI-Hap II) represents the difference in the fraction of total lengthcontributed by short fragments for Hap I and Hap II. The ΔF(Hap I-HapII) values ranged from −0.0203 to 0.0207 with a median of 0.0003. Thesmall ΔF(Hap I-Hap II) value is compatible with the same sizedistribution for fragments mapping to Hap I and Hap II. Thus, we candeduce that the fetus is heterozygous for Hap I and Hap II. As type βSNPs are defined as SNPs at which the paternal alleles are identical tothe maternal alleles on Hap II, this result implies that the fetus hasinherited Hap I from the mother.

FIG. 30 shows a plot of ΔF(Hap I-Hap II) for type α and type β SNPs onchromosome 22 according to embodiments of the present invention. Fortype α SNPs, the size distribution of Hap I fragments is shorter thanthat of Hap II fragments, which results in values of ΔF(Hap I-Hap II)greater than zero. For type β SNPs, there is no difference between thesize distribution of Hap I and Hap II fragments; therefore, the ΔF(HapI-Hap II) values cluster around 0. Using a cutoff of 0.025, a ΔF(HapI-Hap II) analysis can correctly deduced the inheritance of Hap I by thefetus for all the 13 type α segments and 21 type β segments.

XII. Example of Using Targeted Sequencing

The following example demonstrates that a size-based diagnostic approachof embodiments of the present invention can be usable in a targetedsequencing format. In such a format, genomic regions of diagnosticinterest are specifically targeted for the sequencing.

This format has the advantage that the sequencing is focused on theregion of interest, in contrast to the situation involving randomsequencing, where some of the sequencing power is used for regions whichare not immediately relevant to the diagnostic application. Thus, thetargeted sequencing format can be expected to increase the throughputand reduce the cost of the system. Targeted sequencing can be performedusing any format known to those of skill in the art, including asolution-phase capture system (e.g. the Agilent SureSelect system), asolid-phase capture system (e.g. the Roche NimbleGen system) or bytarget-specific amplification (e.g. the RainDance system).

Blood samples were collected from eight pregnant women during theirfirst trimester. DNA from 3.2 mL of plasma was extracted for each caseby the DSP DNA Blood Mini Kit (Qiagen). Karyotyping performed on thechorionic villus samples (collected after maternal blood samples hadbeen taken) indicated that four fetuses had T21 (UK229, UK510, UK807,PW421), while the other four were euploid males (PW226, PW263, PW316,PW370).

5 to 30 ng of plasma DNA for each case was used for DNA libraryconstruction by the paired-end sample preparation kit (Illumina)according to the manufacturer's protocol of ChromatinImmunoprecipitation Sequencing sample preparation. The adapter-ligatedDNA was purified directly using spin columns provided in a QIAquick PCRpurification kit (Qiagen), without further size selection. Theadapter-ligated DNA was then amplified using a 15-cycle PCR withstandard primers. The primers were PCR Primer PE 1.0 and 2.0 fromIllumina. The DNA libraries were quantified by using a NanoDrop ND-1000spectrophotometer (NanoDrop Technologies) and run on a 2100 Bioanalyzer,using a DNA 1000 kit (Agilent), to check for size distribution. 0.6 to 1μg of an amplified plasma DNA library was generated for each sample inan average size of about 290 bp.

The SureSelect Human All Exon capture library was obtained from Agilentand covered 37.8 Mb of human exons (catalog number: 5190-2310). For alleight cases in this study, 500 ng of the amplified plasma DNA library ofeach case was incubated with the capture probes for 24 hours at 65° C.,according to the manufacturer's instruction. After hybridization, thecaptured targets were selected by pulling down the biotinylatedprobe/target hybrids by using streptavidin-coated magnetic beads (DynalDynaMag-2 Invitrogen), and purified with the MinElute PCR PurificationKit (Qiagen). Finally, the targeted DNA libraries were enriched by12-cycle PCR amplification with SureSelect GA PE primers from Agilent.The PCR products were purified by a QIAquick PCR Purification Kit(Qiagen).

Eight pairs of libraries with and without target-enrichment were loadedonto 16 lanes of two flow cells, and then sequenced by a Genome AnalyzerIIx (Illumina) using a 36-bp×2 paired end format. All 36-bp sequencedreads were aligned to the unmasked human reference genome (Hg18)(genome.ucsc.edu), using the Short Oligonucleotide Alignment Program 2(soap.genomics.org.cn/). The fragment size of paired-end reads wasdefined ranging from 40 bp to 600 bp. The size of each sequenced DNAfragment was inferred from the coordinates of the outermost nucleotidesat both ends.

Size Analysis for Prenatal Detection of Fetal Trisomy 21

In this example, fractions of total length contributed by shortfragments were calculated for chromosome 21 and the referencechromosomes, represented by F₂₁ and F_(ref), respectively. The referencechromosomes consisted of all autosomes except chromosomes 13, 18 and 21.The total length was calculated by the summation of length for all DNAfragments of 600 bp or less. A difference in the fraction of lengthcontributed by short fragments between chromosome 21 and the referencechromosomes (ΔF) was calculated as F₂₁−F_(ref).

FIG. 31A is a table that provides a size analysis of plasma DNA withouttarget enrichment according to embodiments of the present invention.Various columns provide total length for fragments ≦150 bp forchromosome 21, total length for fragments ≦600 bp for chromosome 21, andthe ratio of the two as F₂₁. Other columns provide total length forfragments ≦150 bp for the reference chromosomes, total length forfragments ≦600 bp for the reference chromosomes, and the ratio of thetwo as F_(ref). The last column is the difference between the twofractions, ΔF.

FIG. 31B is a table that provides a size analysis of plasma DNA withtarget enrichment according to embodiments of the present invention. Thecolumns of FIG. 31B have the same data format as the table for FIG. 31B.

FIG. 32 is a plot of ΔF for the T21 and euploid samples with and withouttarget enrichment. For the samples without target enrichment, using acutoff value of 0.005 for ΔF, the plasma samples from T21 and euploidpregnancies can be differentiated with 100% accuracy. For the sampleswith target enrichment, using a cutoff value of 0.004 for ΔF, the plasmasamples from T21 and euploid pregnancies can be differentiated with 100%accuracy. This example demonstrates that the size-based analysis can beperformed using targeted sequencing. For the detection of T21, it may beadvantageous to use targeted sequencing for chromosome 21 and areference chromosome, such that 50% of the sequencing is directed to theformer and the rest is directed to the latter. Such a design can reducethe waste of sequencing power to regions that are not immediatelyrelevant to the detection of T21. Such a design can allow samples frommultiple patients to be sequenced using multiplex sequencing (e.g. byusing indexed or barcoded sequencing).

Any of the software components or functions described in thisapplication, may be implemented as software code to be executed by aprocessor using any suitable computer language such as, for example,Java, C++ or Perl using, for example, conventional or object-orientedtechniques. The software code may be stored as a series of instructions,or commands on a computer readable medium for storage and/ortransmission, suitable media include random access memory (RAM), a readonly memory (ROM), a magnetic medium such as a hard-drive or a floppydisk, or an optical medium such as a compact disk (CD) or DVD (digitalversatile disk), flash memory, and the like. The computer readablemedium may be any combination of such storage or transmission devices.

Such programs may also be encoded and transmitted using carrier signalsadapted for transmission via wired, optical, and/or wireless networksconforming to a variety of protocols, including the Internet. As such, acomputer readable medium according to an embodiment of the presentinvention may be created using a data signal encoded with such programs.Computer readable media encoded with the program code may be packagedwith a compatible device or provided separately from other devices(e.g., via Internet download). Any such computer readable medium mayreside on or within a single computer program product (e.g. a hard driveor an entire computer system), and may be present on or within differentcomputer program products within a system or network. A computer systemmay include a monitor, printer, or other suitable display for providingany of the results mentioned herein to a user.

An example of a computer system is shown in FIG. 33. The subsystemsshown in FIG. 33 are interconnected via a system bus 3375. Additionalsubsystems such as a printer 3374, keyboard 3378, fixed disk 3379,monitor 3376, which is coupled to display adapter 3382, and others areshown. Peripherals and input/output (I/O) devices, which couple to I/Ocontroller 3371, can be connected to the computer system by any numberof means known in the art, such as serial port 3377. For example, serialport 3377 or external interface 3381 can be used to connect the computerapparatus to a wide area network such as the Internet, a mouse inputdevice, or a scanner. The interconnection via system bus allows thecentral processor 3373 to communicate with each subsystem and to controlthe execution of instructions from system memory 3372 or the fixed disk3379, as well as the exchange of information between subsystems. Thesystem memory 3372 and/or the fixed disk 3379 may embody a computerreadable medium. Any of the values mentioned herein can be output fromone component to another component and can be output to the user.

A computer system can include a plurality of the same components orsubsystems, e.g., connected together by external interface 3381 or by aninternal interface. In some embodiments, computer systems, subsystem, orapparatuses can communicate over a network. In such instances, onecomputer can be considered a client and another computer a server, whereeach can be part of a same computer system. A client and a server caneach include multiple systems, subsystems, or components.

The specific details of particular embodiments may be combined in anysuitable manner without departing from the spirit and scope ofembodiments of the invention. However, other embodiments of theinvention may be directed to specific embodiments relating to eachindividual aspect, or specific combinations of these individual aspects

The above description of exemplary embodiments of the invention has beenpresented for the purposes of illustration and description. It is notintended to be exhaustive or to limit the invention to the precise formdescribed, and many modifications and variations are possible in lightof the teaching above. The embodiments were chosen and described inorder to best explain the principles of the invention and its practicalapplications to thereby enable others skilled in the art to best utilizethe invention in various embodiments and with various modifications asare suited to the particular use contemplated.

All publications, patents, and patent applications cited herein arehereby incorporated by reference in their entirety for all purposes.

What is claimed is:
 1. A method for performing prenatal diagnosis of asequence imbalance in a biological sample obtained from a female subjectpregnant with a fetus, wherein the biological sample includes nucleicacid molecules that are part of nucleic acid sequences, the biologicalsample including nucleic acid molecules from the fetus and the femalesubject, the method comprising: for each of a plurality of the nucleicacid molecules in the biological sample: measuring a size of the nucleicacid molecule; identifying which nucleic acid sequence the nucleic acidmolecule is derived from; determining, using a computer system, a sizedistribution of the nucleic acid molecules corresponding to a firstsequence; and based on the determined size distribution, determining aclassification of whether a sequence imbalance exists for the firstsequence.
 2. The method of claim 1, further comprising: determining amaternal genotype of the pregnant female subject, wherein the firstsequence contains at least part of the maternal genotype; and based onthe maternal genotype of the pregnant female subject and the determinedsize distribution, determining a classification of whether a sequenceimbalance exists for the first sequence in the biological samplerelative to the maternal genotype.
 3. The method of claim 2, furthercomprising: using the classification to determine a genotype of thefetus.
 4. The method of claim 1, further comprising: determining amaternal haplotype of the pregnant female subject, wherein the firstsequence contains at least part of the maternal haplotype; and based onthe maternal haplotype of the pregnant female subject and the determinedsize distribution, determining a classification of whether a sequenceimbalance exists for the first sequence in the biological samplerelative to the maternal haplotype.
 5. The method of claim 4, furthercomprising: determining a paternal genotype of the father of the fetus;and using the paternal genotype to determine whether the sequenceimbalance being tested is an overrepresentation or anunderrepresentation relative to the maternal haplotype.
 6. The method ofclaim 4, further comprising: using the classification to determine ahaplotype of the fetus.
 7. The method of claim 1, wherein the firstsequence is a chromosome and the sequence imbalance is a fetalchromosomal aneuploidy.
 8. The method of claim 7, wherein the pluralityof the nucleic acid molecules have been pre-selected based on size. 9.The method of claim 8, wherein the plurality of the nucleic acidmolecules are pre-selected to be less than 200 bases or are pre-selectedto be less than 150 bases.
 10. The method of claim 8, wherein theplurality of the nucleic acid molecules are pre-selected into at leasttwo groups, each having a different range of sizes, the method furthercomprising: determining a size distribution of the nucleic acidmolecules of each group corresponding to a first chromosome; comparingthe size distributions; and based on the comparisons, determining aclassification of whether a fetal chromosomal aneuploidy exists for thefirst chromosome.
 11. The method of claim 7, wherein determining a sizedistribution of the nucleic acid molecules corresponding to a firstchromosome includes: for each of a plurality of chromosomes, calculatinga statistical value from the sizes of nucleic acid moleculescorresponding to the chromosome, and wherein based on the determinedsize distribution, determining a classification of whether a fetalchromosomal aneuploidy exists for the first chromosome includes:determining a ranking of the chromosomes based on the statistical value;comparing the determined ranking of the first chromosome to anotherranking of the first chromosome obtained from a reference biologicalsample; and based on the comparison, determining a classification ofwhether a fetal chromosomal aneuploidy exists for the first chromosome.12. The method of claim 7, wherein determining a size distribution ofthe nucleic acid molecules corresponding to a first chromosome includes:calculating a first statistical value from the sizes of nucleic acidmolecules corresponding to the first chromosome; calculating a secondstatistical value from the sizes of nucleic acid molecules correspondingto one or more second chromosomes, and wherein based on the determinedsize distribution, determining a classification of whether a fetalchromosomal aneuploidy exists for the first chromosome includes:determining a separation value between the first statistical value andthe second statistical value; comparing the separation value to one ormore cutoff values; and based on the comparison, determining aclassification of whether a fetal chromosomal aneuploidy exists for thefirst chromosome.
 13. The method of claim 12, wherein the separationvalue is a difference between the first statistical value and the secondstatistical value or a result derived from the difference.
 14. Themethod of claim 12, wherein the first statistical value and the secondstatistical value include an average size of the measured sizes for thenucleic acid molecules corresponding to a respective chromosome.
 15. Themethod of claim 12, wherein the first statistical value and the secondstatistical value respectively include a first sum of the sizes of thecorresponding nucleic acid molecules that have a size below a first sizecutoff.
 16. The method of claim 15, wherein the first statistical valueincludes the first sum divided by a second sum, and wherein the secondsum is of the sizes of all of the corresponding nucleic acid moleculesor is of the sizes of the corresponding nucleic acid molecules that havea size below a second size cutoff, the second size cutoff being largerthan the first size cutoff.
 17. The method of claim 15, wherein thefirst statistical value includes the first sum divided by a second sum,and wherein the second sum is of sizes of the corresponding nucleic acidmolecules that are larger than a second size cutoff.
 18. The method ofclaim 17, wherein the second size cutoff is the first size cutoff, andwherein the second sum is of sizes that are below a third size cutoff,the third size cutoff being larger than the second size cutoff.
 19. Themethod of claim 15, further comprising: repeating calculating the firststatistical value and the second statistical value for a plurality offirst size cutoffs, and determining a separation value between the firstand second statistical values for each of the first size cutoff; andidentifying an extremum value of the separation values.
 20. The methodof claim 19, further comprising: comparing the extremum value to the oneor more cutoff values.
 21. The method of claim 19, further comprising:identifying the first size cutoff corresponding to the extremum value;and comparing the first size cutoff corresponding to the extremum valueto the one or more cutoff values.
 22. The method according to claim 7,where the biological sample includes blood, plasma, serum, maternalblood containing fetal cells, fetal cells obtained from maternal blood,urine, saliva, or uterine lavage fluid.
 23. A computer program productcomprising a non-transitory computer readable medium storing a pluralityof instructions for controlling a processor to perform an operation forperforming prenatal diagnosis of a sequence imbalance in a biologicalsample obtained from a female subject pregnant with a fetus, wherein thebiological sample includes nucleic acid molecules that are part ofnucleic acid sequences, the biological sample including nucleic acidmolecules from the fetus and the female subject, the instructionscomprising: for each of a plurality of the nucleic acid molecules in thebiological sample: measuring a size of the nucleic acid molecule;identifying which nucleic acid sequence the nucleic acid molecule isderived from; determining a size distribution of the nucleic acidmolecules corresponding to a first sequence; and based on the determinedsize distribution, determining a classification of whether a sequenceimbalance exists for the first sequence.
 24. A method for performingprenatal diagnosis of a fetal chromosomal aneuploidy in a biologicalsample obtained from a female subject pregnant with a fetus, wherein thebiological sample includes nucleic acid molecules from the fetus and thefemale subject, the method comprising: for each of a plurality of thenucleic acid molecules in the biological sample: measuring a size of thenucleic acid molecule; identifying which chromosome the nucleic acidmolecule is derived from; for each of a plurality of chromosomes,calculating, using a computer system, a statistical value from the sizesof nucleic acid molecules corresponding to the chromosome; determining aranking of the chromosomes based on the statistical value; comparing thedetermined ranking of a first chromosome to another ranking of the firstchromosome obtained from a reference biological sample; and based on thecomparison, determining a classification of whether a fetal chromosomalaneuploidy exists for the first chromosome.
 25. The method of claim 24,wherein comparing the determined ranking of a first chromosome toanother ranking of the first chromosome obtained from a referencebiological sample includes: determining a first difference between thedetermined ranking of the chromosome and a ranking of a referencechromosome for the biological sample; determining a second differencebetween the another ranking of the chromosome and a ranking of thereference chromosome for the reference biological sample; and comparingthe differences.
 26. The method of claim 24, wherein measuring the sizeof each of the plurality of nucleic acid molecules includes: receivingthe biological sample; and sequencing at least a portion of a pluralityof the nucleic acid molecules contained in the biological sample. 27.The method of claim 26, wherein the sequenced portion of each nucleicacid molecule includes both ends of the respective nucleic acidmolecule.
 28. The method of claim 24, wherein the statistical valueincludes the median size of the measured sizes for the nucleic acidmolecules corresponding to a respective chromosome.
 29. The method ofclaim 24, wherein the statistical value includes an average size of themeasured sizes for the nucleic acid molecules corresponding to arespective chromosome.
 30. The method of claim 24, wherein the measuredsize is a length or a molecular mass or a measured parameter that isrelated to the length.
 31. The method of claim 30, wherein the measuredsize is a fluorescence intensity that corresponds to the length.
 32. Themethod of claim 24, wherein the ranking is lowest to highest in thestatistical values.
 33. The method of claim 24, wherein the ranking ishighest to lowest in the statistical values.
 34. A computer programproduct comprising a non-transitory computer readable medium storing aplurality of instructions for controlling a processor to perform anoperation for performing prenatal diagnosis of a fetal chromosomalaneuploidy in a biological sample obtained from a female subjectpregnant with a fetus, wherein the biological sample includes nucleicacid molecules from the fetus and the female subject, the instructionscomprising: for each of a plurality of the nucleic acid molecules in thebiological sample: measuring a size of the nucleic acid molecule;identifying which chromosome the nucleic acid molecule is derived from;for each of a plurality of chromosomes, calculating a statistical valuefrom the sizes of nucleic acid molecules corresponding to thechromosome; determining a ranking of the chromosomes based on thestatistical value; comparing the determined ranking of a firstchromosome to another ranking of the first chromosome obtained from areference biological sample; and based on the comparison, determining aclassification of whether a fetal chromosomal aneuploidy exists for thefirst chromosome.
 35. A method for performing prenatal diagnosis of afetal chromosomal aneuploidy in a biological sample obtained from afemale subject pregnant with a fetus, wherein the biological sampleincludes nucleic acid molecules from the fetus and the female subject,the method comprising: for each of a plurality of the nucleic acidmolecules in the biological sample: measuring a size of the nucleic acidmolecule; identifying which chromosome the nucleic acid molecule isderived from; calculating, using a computer system, a first statisticalvalue from the sizes of nucleic acid molecules corresponding to a firstchromosome; calculating, using the computer system, a second statisticalvalue from the sizes of nucleic acid molecules corresponding to one ormore second chromosomes; determining a separation value between thefirst statistical value and the second statistical value; comparing theseparation value to one or more cutoff values; and based on thecomparison, determining a classification of whether a fetal chromosomalaneuploidy exists for the first chromosome.
 36. The method of claim 35,wherein the separation value is a difference between the firststatistical value and the second statistical value or a result derivedfrom the difference.
 37. The method of claim 35, wherein the separationvalue is a ratio of the first statistical value and the secondstatistical value or a result derived from the ratio.
 38. The method ofclaim 35, wherein the first statistical value and the second statisticalvalue include an average size of the measured sizes for the nucleic acidmolecules corresponding to a respective chromosome.
 39. The method ofclaim 35, wherein the first statistical value and the second statisticalvalue respectively include a first sum of the sizes of the correspondingnucleic acid molecules that have a size below a first size cutoff. 40.The method of claim 39, wherein the first statistical value includes thefirst sum divided by a second sum, and wherein the second sum is of thesizes of all of the corresponding nucleic acid molecules or is of thesizes of the corresponding nucleic acid molecules that have a size belowa second size cutoff, the second size cutoff being larger than the firstsize cutoff.
 41. The method of claim 39, wherein the first statisticalvalue includes the first sum divided by a second sum, and wherein thesecond sum is of sizes of the corresponding nucleic acid molecules thatare larger than a second size cutoff.
 42. The method of claim 39,further comprising: repeating calculating the first statistical valueand the second statistical value for a plurality of first size cutoff,and determining a separation value between the first and secondstatistical values for each of the first size cutoff; and identifying anextremum value of the separation values.
 43. The method of claim 42,further comprising: comparing the extremum value to the one or morecutoff values.
 44. The method of claim 42, further comprising:identifying the first size cutoff corresponding to the extremum value;and comparing the first size cutoff corresponding to the extremum valueto the one or more cutoff values.
 45. The method of claim 35, whereinthe one or more cutoff values are based on a measurement of a percentageof fetal DNA in the biological sample.
 46. A computer program productcomprising a non-transitory computer readable medium storing a pluralityof instructions for controlling a processor to perform an operation forperforming prenatal diagnosis of a fetal chromosomal aneuploidy in abiological sample obtained from a female subject pregnant with a fetus,wherein the biological sample includes nucleic acid molecules from thefetus and the female subject, the instructions comprising: for each of aplurality of the nucleic acid molecules in the biological sample:measuring a size of the nucleic acid molecule; identifying whichchromosome the nucleic acid molecule is derived from; calculating afirst statistical value from the sizes of nucleic acid moleculescorresponding to a first chromosome; calculating a second statisticalvalue from the sizes of nucleic acid molecules corresponding to one ormore second chromosomes; determining a separation value between thefirst statistical value and the second statistical value; comparing theseparation value to one or more cutoff values; and based on thecomparison, determining a classification of whether a fetal chromosomalaneuploidy exists for the first chromosome.